Hormonal and acid-base regulation of phosphoenolpyruvate carboxykinase mRNA levels in rat kidney

Metabolic acidosis (6 days NH4Cl) causes a fourfold increase in the relative abundance of mRNA encoding phosphoenolpyruvate carboxykinase in rat kidney. Streptozotocin-diabetes (6 days) also results in an increased abundance of the mRNA but this increase can be prevented if the acidosis associated with bicarbonate is corrected by treatment with bicarbonate. The results confirm that renal phosphoenolpyruvate carboxykinase is regulated primarily by changes in acid-base status and that this control is at a pretranslational step. Isolated kidney tubules in short-term incubation have been used to identify which agents regulate levels of phosphoenolpyruvate carboxykinase mRNA. The relative abundance of the mRNA was increased by glucocorticoids and hormones which act via cAMP, or by cAMP analogues directly, but was not affected by hormones acting via Ca2+. Neither incubation at pH 7.1 nor the presence of serum from acidotic rats had any effect on the level of phosphoenolpyruvate carboxykinase mRNA. It is concluded that acidosis, glucocorticoids, and cAMP independently regulate expression of renal phosphoenolpyruvate carboxykinase.     

Endogenous and exogenous glucocorticoid regulation of ENaC mRNA expression in developing kidney and lung

Lung liquid absorption at birthis crucial for the successful onset of respiration. Na absorption bythe renal collecting duct plays an important role in renal fluid andelectrolyte homeostasis during the early postnatal period. Theepithelial Na channel (ENaC) plays a central role in mediating thesefunctions, and its subunit expression is developmentally regulated in atemporal and tissue specific pattern. Several lines of evidence suggestthat the prenatal increase in circulating glucocorticoids may play animportant role in increasing ENaC expression during maturation. Wetested the role of the prenatal surge using corticotropin-releasinghormone (CRH) knockout (KO) mice. Relative ENaC expression in lungs of KO mice increased at the same rate as in wild-type (WT) mice, butabsolute expression was only 20-30% of WT. In contrast, relative and absolute expression of all three subunits in kidneys was not different between KO and WT mice. Dexamethasone (Dex) increased -ENaC mRNA in fetal lung and kidney explants within 24 h but had different effects on - or -ENaC. Dex increased - and-ENaC in lung, but only after >48 h of exposure, and had no effecton kidney. The results suggest that the kidney metabolizes endogenous glucocorticoids, but the lung does not. Furthermore, the marked difference between lung and kidney responsiveness to glucocorticoids in- and -ENaC expression suggests that factors other than steroids may be important in regulating functional ENaC expression during development.

     

Divergent regulation of gonadotropin subunit mRNA levels by androgens in the female rat.

To examine the effects of gonadal steroids on the pretranslational regulation of the gonadotropin subunits in the female, adult female rats, beginning 7 or 28 days after ovariectomy, received daily injections of testosterone propionate (T), dihydrotestosterone propionate (D), or estradiol benzoate (E) for 7 days. Intact cycling females and ovariectomized rats that received vehicle served as controls. Serum was obtained for LH and FSH levels to assess changes in gonadotropin secretion. Total RNA from individual rats was recovered and analyzed by blot hybridization with specific radiolabeled cDNA probes for the alpha, LH beta, and FSH beta subunits. Autoradiographic bands were quantitated and standardized to mRNA levels in the intact animals. Ovariectomy resulted in a rise in serum gonadotropin levels and all three gonadotropin subunit mRNA levels. Estrogen replacement resulted in suppression of alpha, LH beta, and FSH beta mRNAs whether given at 7 or 28 days after ovariectomy. In contrast, whereas androgen replacement decreased alpha and LH beta mRNAs, D or T did not consistently suppress FSH beta mRNAs. We conclude that chronic estrogen administration to the castrated female rat uniformly suppresses all three gonadotropin subunit mRNA levels. In female rats, as in male rats, chronic androgen administration fails to negatively regulate FSH beta mRNAs.     

Regulation of ENaC trafficking in rat kidney

The epithelial Na channel (ENaC) forms a pathway for Na⁺ reabsorption in the distal nephron, and regulation of these channels is essential for salt homeostasis. In the rat kidney, ENaC subunits reached the plasma membrane in both immature and fully processed forms, the latter defined by either endoglycosidase H–insensitive glycosylation or proteolytic cleavage. Animals adapted to a low-salt diet have increased ENaC surface expression that is specific for the mature forms of the subunit proteins and is similar (three- to fourfold) for α, β, and γENaC. Kidney membranes were fractionated using differential centrifugation, sucrose-gradient separation, and immunoabsorption. Endoplasmic reticulum membranes, isolated using an antibody against calnexin, expressed immature γENaC, and the content decreased with Na depletion. Golgi membranes, isolated with an antibody against the cis-Golgi protein GM130, expressed both immature and processed γENaC; Na depletion increased the content of processed γENaC in this fraction by 3.8-fold. An endosomal compartment isolated using an antibody against Rab11 contained both immature and processed γENaC; the content of processed subunit increased 2.4-fold with Na depletion. Finally, we assessed the content of γENaC in the late endocytic compartments indirectly using urinary exosomes. All of the γENaC in these exosomes was in the fully cleaved form, and its content increased by 4.5-fold with Na depletion. These results imply that stimulation of ENaC surface expression results at least in part from increased rates of formation of fully processed subunits in the Golgi and subsequent trafficking to the apical membrane.     

Developmental regulation of insulin-like growth factor II mRNA in different rat tissues

Insulin-like growth factor II (IGF-II) is present at high levels in fetal and early neonatal rat plasma, and decreases profoundly following birth. In the present study, the levels of IGF-II RNA in different rat tissues at different ages were determined by hybridization to a rat IGF-II cDNA probe. IGF-II RNA was present in 11 of 13 fetal or neonatal tissues examined: at higher levels in muscle, skin, lung, liver, intestine, and thymus; at lower levels in brain stem, heart, cerebral cortex, kidney, and hypothalamus; and undetectable in spleen and pancreas (although the latter RNA was partially degraded). In each tissue, Northern blot hybridization revealed the presence of six IGF-II RNAs: 6, 4, 3.8, 2.2, 1.7, and 1.2 kilobase pairs, consistent with results previously observed in the BRL-3A rat liver cell line and attributed to alternative RNA processing. Although differences in the relative abundance of these RNAs were observed in different tissues, the same size species occurred in all tissues with the 4-kilobase pair RNA the most abundant species. RNAs from the different tissues were examined at six developmental ages (days 16 and 21 of gestation; days 2, 11, 22, and 75 after birth) by hybridization to slot blots and Northern blots. In lung, thymus, kidney, and brain stem, IGF-II RNA was expressed at higher levels in the fetus than after birth, whereas in muscle, skin, liver, heart, and intestine, the high fetal levels of IGF-II RNA continued through day 11 or day 22 after birth. IGF-II RNA persisted into adulthood in cerebral cortex and hypothalamus. Although the significance of these tissue-specific differences in the developmental regulation of the expression of IGF-II RNA remains to be established, they exhibit intriguing temporal correlations with major maturational events in some tissues such as lung and muscle.     

Developmental expression of the GABAA receptor alpha 1 subunit mRNA in the rat brain

Recent studies have suggested that the GABAA, receptor complex, the site of action of the inhibitory neurotransmitter gamma amino-butyric acid (GABAA) and the anxiolytic benzodiazepines, is heterogeneous. Moreover, its composition may change during development. To better understand the molecular basis of receptor heterogeneity, the levels and distribution of the mRNA encoding the alpha 1 receptor subunit were examined in the developing and adult rat brain with quantitative in situ hybridization histochemistry. Our studies demonstrate that alpha 1 subunit mRNA expression changes during ontogeny. At late embryonic stages and in the first postnatal week, low levels of the mRNA were detected in the cortex, inferior colliculus, and hippocampus. The mRNA levels in these regions increased during the second and third postnatal weeks. Furthermore, a dramatic change in the distribution of the alpha 1 subunit mRNA was seen in the second postnatal week when the message first became detectable in the cerebellar cortex. During subsequent development and in the mature brain, the alpha 1 subunit mRNA was most abundant in the cerebellum, olfactory bulb, and inferior colliculus, although the absolute levels of mRNA varied by as much as sixfold in selected brain regions. The mature distribution of alpha 1 subunit mRNA, along with its temporal appearance in the cerebellum, suggests that this subunit is a constituent of the Type 1 benzodiazepine site of the GABAA receptor complex. Furthermore, the onset of alpha 1 subunit mRNA expression in the cerebellar cortex coincides with a period of extensive synapse formation, raising the possibility that synaptic interactions modulate the appearance of this GABAA receptor subunit in the cerebellum.     

Developmental and growth-related regulation of expression of serine dehydratase mRNA in rat liver

In rat liver, serine dehydratase mRNA is undetectable in the late prenatal period, but its level increases rapidly after birth to a transient peak, and then after decrease gradually increases again to a maximum 2 weeks after birth that is slightly higher than that of adult liver. To determine whether mature quiescent hepatocytes proliferate without loss of differentiated functions, we measured the serine dehydratase mRNA contents in regenerating liver and primary cultured hepatocytes from adult rats. Partial hepatectomy resulted in a dramatic decrease in the mRNA content within 24 h and then its recovery within a week. In subconfluent cultures of adult rat hepatocytes that did not grow even in the presence of mitogens, serine dehydratase mRNA was maintained at a high level. However, when the hepatocytes were cultured at low cell density without added mitogens, their serine dehydratase mRNA content decreases to a quarter of that of subconfluent cultures. The possibility that the expression of serine dehydratase mRNA is regulated in G0/G1 transition before entry into the S phase and the relationship of the mRNA with growth are discussed.     

Hormonal regulation of nuclear cyclic AMP-dependent protein kinase subunit levels in rat ovaries

Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear cAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity labeling of nuclear extracts with 8-azido-[32P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estrogen-primed hypophysectomized rats. After follicle-stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, Mr = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity. Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density. These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.     

Selenium regulation of thioredoxin reductase activity and mRNA levels in rat liver

Mammalian thioredoxin reductase (TRR; NADPH₂:oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) is a new member of the family of selenocysteine-containing proteins. TRR activity in Se-deficient rat liver is reported to decrease to 4.5 to 15% of the activity in Se-adequate rat liver, similar to the fall in Se-dependent glutathione peroxidase-1 activity. Both glutathione peroxidase-1 enzyme activity and mRNA levels decrease dramatically in Se deficiency, whereas glutathione peroxidase-4 activity only decreases to 40% of Se-adequate levels and mRNA level is little affected by Se deficiency. The purpose of these experiments is to study the effect of Se status on TRR mRNA levels and enzyme activity in our well-characterized rat model, and to compare this regulation directly to the regulation of other Se-dependent proteins in male weanling rats fed Se-deficient diets or supplemented with dietary Se for 28 days. In two experiments, TRR activity in Se-deficient liver decreased to 15% of Se-adequate activity as compared to 2% and 40% of Se-adequate levels for GPX1 and GPX4, respectively. Using ribonuclease protection analysis, we found that TRR mRNA levels in Se-deficient rat liver decreased to 70% of Se-adequate levels. This decrease in TRR mRNA was similar to the GPX4 mRNA decrease in Se-deficient liver in these experiments, whereas GPX1 mRNA levels decreased to 23% of Se-adequate levels. This study clearly shows that TRR represents a third pattern of Se regulation with dramatic down-regulation of enzyme activity in Se deficiency but with only a modest decrease in mRNA level. The conservation of TRR mRNA in Se deficiency suggests that this is a valued enzyme; the loss of TRR activity in Se deficiency may be the cause of some signs of Se deficiency.     

Developmental regulation of hormone-sensitive lipase mRNA in the rat: changes in steroidogenic tissues.

The hydrolysis of triglycerides and cholesteryl esters stored within cells is mediated by the enzyme, hormone-sensitive lipase. In adipose tissue and heart, hormone-sensitive lipase primarily hydrolyzes stored triglycerides to free fatty acids, while in steroidogenic tissues, it principally converts cholesteryl esters to free cholesterol for steroid hormone production. To determine whether hormone-sensitive lipase is under tissue-specific, developmental regulation, the steady state levels of hormone-sensitive lipase mRNA were determined in normal rats from late fetal life through 2 years of age. Hormone-sensitive lipase mRNA levels did not appear to vary in adipose tissue from epididymal fat pads obtained from animals between 3 weeks and 2 years of age. In heart, hormone-sensitive lipase mRNA levels were lowest in the fetus increased rapidly within the first day postnatally, and then gradually increased to stable adult levels by 2 months that were 3-fold higher than observed in fetal rats. Steady state mRNA levels of hormone-sensitive lipase in the adrenals were lowest in fetal rats, increased 4-fold during the first day and peaked at levels that were 9-fold higher by the end of the first week. Thereafter, levels fell and remained 3- to 4-fold higher than at birth throughout adult life. Hormone-sensitive lipase mRNA was undetectable in testes before 4 weeks of age and increased 25-fold to stable adult levels between 4 and 12 weeks. Thus, hormone-sensitive lipase is differentially expressed and regulated in a tissue-specific fashion during development and aging.     

Developmental regulation of NMDA receptor 2B subunit mRNA and ifenprodil binding in the zebra finch anterior forebrain

In passerine songbirds, song learning often is restricted to an early sensitive period and requires the participation of several discrete regions within the anterior forebrain. Activation of N-methyl-D-aspartate (NMDA) receptors is implicated in song learning and in one forebrain song region, the lateral magnocellular nucleus of the anterior neostriatum (IMAN), NMDA receptors decrease in density, their affinity for the antagonist MK-801 increases, and their currents decay more quickly as young male zebra finches lose the ability to imitate new song elements. These developmental changes in NMDA receptor pharmacology and physiology suggest that the subunit composition of NMDA receptors changes developmentally. Here, we have used in situ hybridization and [3H]ifenprodil receptor autoradiography to study the developmental regulation of the NMDA receptor 2B subunit (NR2B) within the anterior forebrain of male zebra finches. NR2B mRNA expression within the IMAN was twice as great in 30-day-old males (early in the sensitive period for song learning) as in adult males, and this developmental decrease in NR2B mRNA expression was mirrored by a decrease in high-affinity (NR2B-associated) [3H]ifenprodil binding within this song region. In another anterior forebrain song region, Area X, NR2B mRNA also declined significantly after 30 days posthatch, but this decline was not accompanied by a significant decrease in [3H]ifenprodil binding. The results are consistent with the hypothesis that developmental changes in NMDA receptor function mediated by regulation of subunit composition contribute to the sensitive period for vocal learning in birds.     

Aldosterone-induced abnormal regulation of ENaC and SGK1 in Dahl salt-sensitive rat

Aldosterone plays a crucial role in controlling mineral balance in our body. The mechanism of aldosterone has been reported to elevate renal Na+ reabsorption by stimulating expression of epithelial Na+ channel (ENaC) and also activate an ENaC-regulating protein kinase, serum and glucocorticoid-regulated kinase 1 (SGK1). However, it is unknown whether aldosterone shows its stimulatory action on ENaC and SGK1 under an abnormal, salt-sensitive hypertensive condition. To clarify this point, we studied how aldosterone regulates expression of ENaC and SGK1 in Dahl salt-sensitive (DS) rat that shows hypertension with high salt diet. RNA and protein were extracted from the kidney 6 h after application of aldosterone (1.5 mg/kg body weight) subcutaneously injected into adrenalectomized DS and Dahl salt-resistant (DR) rats. Aldosterone decreased mRNA expression of beta- and gamma-ENaC in DS rat unlike DR rat, while aldosterone increased alpha-ENaC mRNA expression in DS rat similar to DR rat. Further, we found that aldosterone elevated SGK1 expression in DR rat, but not in DS rat. These observations indicate that ENaC and SGK1 are abnormally regulated by aldosterone in salt-sensitive hypertensive rats, suggesting that disturbance of the aldosterone regulation would be one of factors causing salt-sensitive hypertension.     

Developmental regulation of metallothionein mRNA, zinc and copper levels in rainbow trout, Salmo gairdneri

The metallothionein (MT) gene expression profile was followed in rainbow trout during early embryo development and in liver and gonads during the period of sexual maturation. The hepatic MT mRNA levels increase at the end of sexual maturation in both male and female rainbow trout. Although both isoforms of MT mRNA accumulate in the liver, there is a preferential increase in MT-A in the female liver. Concomitantly with this increase in MT there is a redistribution of zinc and copper to MT. In the juvenile female there is an abundance of MT mRNA in the ovaries. This is correlated to high levels of zinc in the MT fraction upon Sephadex G-75 chromatography. During ovary development the MT mRNA levels and the MT-bound zinc levels drop, with an increase in zinc being bound to high-molecular-mass proteins. At ovulation most of the zinc is found in the membrane portion upon centrifugation. In contrast to the ovaries, there are no apparent changes in either trace metal distribution or MT mRNA levels during testis development. In the developing embryo there is an increase in MT-bound copper at gastrulation. This is accompanied by an increase in both isoforms of MT mRNA. At hatch both the copper and zinc levels increase in the MT fraction, with a concomitant increase in mainly MT-A mRNA. These findings indicate that the variations in MT mRNA levels during development are closely associated with metal regulation.     

Flavonoid-induced reduction of ENaC expression in the kidney of Dahl salt-sensitive hypertensive rat

Flavonoid, a plant extract, exhibits various biological actions. Dietary flavonoid intake is reported to reduce an elevated blood pressure, however the mechanism is unknown. The epithelial Na+ channel (ENaC) in the kidney plays a key role in the regulation of blood pressure by contributing to the Na+ reabsorption in renal tubules. Thus, we investigated the effect of quercetin, a flavonoid, on ENaC mRNA expression in the kidney of hypertensive Dahl salt-sensitive rats. Dahl salt-sensitive rats of 8 weeks were acclimated for 1 week in a metabolic cage and were subsequently kept for 4 weeks under four different conditions: (1) normal salt diet (0.3% NaCl), (2) normal salt diet with quercetin (10 mg/kg/day), (3) high-salt diet (8% NaCl), and (4) high-salt diet with quercetin. Quercetin diminished the alphaENaC mRNA expression in the kidney associated with reduction of the systolic blood pressure elevated by high-salt diet, suggesting that one of the mechanisms of the flavonoid's antihypertensive effect on salt-sensitive hypertension would be mediated through downregulation of ENaC expression in the kidney.     

Regulation of ornithine aminotransferase mRNA levels in rat kidney by estrogen and thyroid hormone

The regulation of the mitochondrial matrix enzyme, ornithine aminotransferase, by estrogen and triiodothyronine (T3) in rat kidney was examined using a cloned cDNA probe and in vitro translation of poly(A+) RNA. After a single, acute dose of either 17 beta-estradiol or T3, the rate of enzyme synthesis and the levels of translatable and hybridizable ornithine aminotransferase mRNA all increase in parallel. Levels of hybridizable mRNA were estimated by hybridization of randomly 32P-labeled RNA to filter-bound plasmid DNA. Maximal levels of induction by estrogen and T3 were about 15- and 3-fold, respectively. Lag times of at least 5 h and less than 3 h were observed for induction by T3 and estrogen. T3 and estrogen exert a synergistic effect in increasing ornithine aminotransferase mRNA levels. 16 h after T3 administration and 24 h after estrogen administration, a 1.6- and 13-fold increase in mRNA levels were observed. Both of these treatments in combination for the indicated time periods resulted in a 21-fold increase in ornithine aminotransferase mRNA. From the mRNA accumulation curves, half-lives of 10 to 14 h and 12 to 16 h were estimated for the mRNA after estrogen and T3 induction, respectively. These similar half-lives suggest that an increase in the rate of mRNA production is primarily responsible for the induction observed after estrogen administration.     

Developmental regulation of insulin-like growth factor-II/mannose 6-phosphate receptor mRNA in the rat.

This study examined levels of insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6PR) mRNA in tissues of rats at different stages of growth. Northern blot analysis of total RNA from tissues of rats aged 2, 9, 21 and 42 days and from 21 day fetal rats was carried out using a cDNA probe to the IGF-II/M6PR. Northern blots showed this probe hybridized to a single 9kb band in all tissues tested. Highest hybridization signals were detected in fetal and neonatal tissues with levels rapidly decreasing after birth. For all age groups tested the highest signal was obtained with heart followed by muscle, lung, and kidney, with liver and brain showing lower levels of message. These results indicate that IGF-II/M6PR mRNA is developmentally regulated, and suggest a role for the IGF-II/M6PR in fetal and neonatal growth.     

Developmental expression of the GABAA receptor α1 subunit mRNA in the rat brain

Recent studies have suggested that the GABA_A, receptor complex, the site of action of the inhibitory neurotransmitter gamma amino-butyric acid (GABA_A) and the anxiolytic benzodiazepines, is heterogeneous. Moreover, its composition may change during development. To better understand the molecular basis of receptor heterogeneity, the levels and distribution of the mRNA encoding the α1 receptor subunit were examined in the developing and adult rat brain with quantitative in situ hybridization histochemistry. Our studies demonstrate that α1 subunit mRNA expression changes during ontogeny. At late embryonic stages and in the first postnatal week, low levels of the mRNA were detected in the cortex, inferior colliculus, and hippocampus. The mRNA levels in these regions increased during the second and third postnatal weeks. Furthermore, a dramatic change in the distribution of the α1 subunit mRNA was seen in the second postnatal week when the message first became detectable in the cerebellar cortex. During subsequent development and in the mature brain, the α1 subunit mRNA was most abundant in the cerebellum, olfactory bulb, and inferior colliculus, although the absolute levels of mRNA varied by as much as sixfold in selected brain regions. The mature distribution of α1 subunit mRNA, along with its temporal appearance in the cerebellum, suggests that this subunit is a constituent of the Type 1 benzodiazepine site of the GABA_A receptor complex. Furthermore, the onset of α1 subunit mRNA expression in the cerebellar cortex coincides with a period of extensive synapse formation, raising the possibility that synaptic interactions modulate the appearance of this GABA_A receptor subunit in the cerebellum.     

Hormonal regulation of tissue plasminogen activator secretion and mRNA levels in rat granulosa cells

Granulosa cells from immature rats produce tissue plasminogen activator (tPA) in response to follicle stimulating hormone (FSH) or luteinizing hormone (LH) both in vitro and in vivo. We have used the in vitro system to investigate the level at which the hormonal induction of tPA is regulated. Within 12 h following FSH addition, a dramatic but transient increase in tPA secretion occurs for by 24 h secretion returns to basal levels. This pattern of enzyme induction is similar with LH, but the onset of the increase is delayed. When steady-state tPA mRNA levels are examined after hormone treatment, the results mirror those obtained if one measures enzyme activity; a large increase in tPA mRNA followed by a decrease to basal levels is observed with both hormones, and the lag in induction by LH is also apparent. These results demonstrate that the regulation of tPA activity by gonadotropins occurs at the level of the steady-state concentration of the mRNA. In the presence of cycloheximide, the induction of tPA mRNA by FSH or LH is not greatly affected, indicating that this phase of the response to gonadotropins does not require the synthesis of new protein. However, the decrease in tPA mRNA levels observed 24 h after FSH treatment is affected by cycloheximide, in that the drug delays the reduction in mRNA levels seen with hormone alone.