Differential expression of the multiple forms of rat prekininogen mRNAs after acute inflammation

Responses of the rat liver prekininogen mRNAs after induction of acute inflammation were examined by blot-hybridization and S1 nuclease protection analyses with the aid of cDNA probes specific for rat kininogens. Marked changes in the relative levels of the low molecular weight (LMW) prekininogen mRNAs were observed after administration of Escherichia coli lipopolysaccharide, and the mRNA levels increased with a half-maximal dose of approximately 100 ng of lipopolysaccharide/100 g body weight. At maximum level of induction, the LMW prekininogen mRNAs comprised about 1% of total liver mRNA, thus representing a major component of the liver mRNA in the acutely inflamed rat. Differences in the inflammatory responses of various forms of the prekininogen mRNAs were then investigated by S1 nuclease protection analysis with the use of three different cDNA probes, each specific for either K-prekininogen or two types of T-prekininogens. Both of the T-prekininogen mRNAs increased progressively during the first 24 h after induction of inflammation, and at maximum level of induction, these two mRNAs increased about 10- and 13-fold over their normal level. In contrast, neither of the high molecular weight and LMW K-prekininogen mRNAs exhibited such an increase after induction of inflammation. Thus, the expressions of the rat T- and K-prekininogen mRNAs are differentially regulated in response to the induction of acute inflammation.     

Tissue distribution of rat angiotensinogen mRNA and structural analysis of its heterogeneity

The tissue distribution and the structural heterogeneity of the rat angiotensinogen mRNA have been investigated with the aid of a previously cloned cDNA as well as a genomic DNA for rat angiotensinogen as analytical probes. The angiotensinogen mRNA is expressed not only in the liver but also in various tissues including the brain, kidney, adrenal gland, ovary, and lung. The relative levels of the mRNA in the above tissues have been estimated to be 3-4, 20-30 (for the next three tissues), and around 100 times less than that in the liver, respectively. The mRNAs in both hepatic and extrahepatic tissues are encoded by a single gene in the rat genome. At least four different size classes of the angiotensinogen mRNA that start with a single 5' terminus and differ only in the lengths of their 3'-untranslated regions have been identified, and these multiple mRNA species are most likely generated by using the polyadenylation signals AAUAAA and AUUAAA found 10-30 nucleotides upstream from the four polyadenylation sites. Because the structures of these multiple mRNA species do not vary among the tissues of the liver, brain, and kidney, angiotensinogen synthesized locally is structurally identical to that produced in the liver and may have some biological roles independent of the circulating angiotensinogen, mainly derived from the liver. In addition, the sequence of the 5'-flanking region of the angiotensinogen gene has been determined, and some features common to other steroid hormone-responsive genes have been discussed.     

Tissue-specific regulation of angiotensinogen mRNA accumulation by dexamethasone

The regulation of angiotensinogen gene expression in response to adrenalectomy and dexamethasone treatment was examined in multiple rat tissues. Angiotensinogen mRNA as quantitated by slot blot hybridization utilizing an angiotensinogen cRNA probe was most abundant in the liver with levels in the brain, kidney, and adrenal of 50, 25, and 10%, respectively. No angiotensinogen mRNA was detected in testes or heart. Although no change in the quantity of angiotensinogen mRNA was found following adrenalectomy and maintenance on 0.9% saline, dexamethasone treatment of both normal and adrenalectomized rats resulted in a time-dependent and tissue-specific accumulation of angiotensinogen mRNA. In normal animals, the hepatic response to treatment was a 4.5-fold increase in angiotensinogen mRNA by 8 h which remained 2.4-fold above basal levels by 24 h. Angiotensinogen mRNA levels in the brains of normal rats treated with dexamethasone increased only 60% by 6 h and returned to basal levels by 24 h. In contrast to the increases seen in brain and liver, angiotensinogen mRNA derived from kidney did not significantly change following dexamethasone treatment. In adrenalectomized animals, the hepatic response to dexamethasone was similar to normal animals with a 3.7-fold increase by 6 h. The accumulation in brain was greater in these animals compared to normals and increased 3-fold by 8 h. Finally, dexamethasone did not significantly increase levels in the kidney. These results clearly demonstrate glucocorticoid regulation of angiotensinogen mRNA levels in liver and brain. In contrast, the kidney, an organ known to contain glucocorticoid receptors, does not respond with increased angiotensinogen mRNA levels following glucocorticoid stimulation. These studies provide the first evidence for tissue-specific differences in the control of angiotensinogen mRNA.     

Immunochemical studies on biosynthesis of rat plasma angiotensinogen and its regulation by cortisol

Glucocorticosteroid hormones increase the level of rat plasma angiotensinogen by increasing its rate of synthesis. Two forms of plasma angiotensinogen have been purified differing with respect to molecular weight and affinity to concanavalin A. Immunochemical studies using antibodies raised against the separated forms of angiotensinogen revealed cross-reactivity with both antigens. Both antibodies were able to quantitatively precipitate the angiotensinogen activity present in rat serum samples. Cortisol increased the total amount of plasma renin substrate without changing the relative amounts of both angiotensinogen forms. mRNA coding for plasma angiotensinogen was determined by in vitro translation of poly(A)-containing RNA and immunochemical analysis of translation products. Angiotensinogen mRNA could be detected in total poly(A)-containing RNA isolated from rat liver, but not in mRNA isolated from brain, although angiotensinogen has been reported to be present in the latter organ. The level of hepatic mRNA coding for plasma angiotensinogen was high in rats treated with cortisol, but not detectable in animals depleted from endogenous glucocorticosteroids by bilateral adrenalectomy.     

Tissue specific hormonal regulation of the rat angiotensinogen gene expression

Angiotensinogen is the precursor of the most potent pressor substance, angiotensin. Angiotensinogen levels are increased in some forms of human hypertension. Its levels are modulated by various factors including glucocorticoids, estrogens, and prostaglandins. We have recently reported the isolation of a human angiotensinogen cDNA clone and provided evidence for the presence of its mRNA in rat liver, brain, and heart. In this communication we report the effect of dexamethasone and estradiol on angiotensinogen mRNA levels in rat liver, brain, and heart. Our results indicate that angiotensinogen levels are increased to different extents in these three tissues as a result of glucocorticoid or estrogen administration.     

Glucose-dependent regulation of glucose transport activity, protein, and mRNA in primary cultures of rat brain glial cells

D-Glucose deprivation of primary rat brain glial cell cultures, by incubation with 25 mM D-fructose for 24 h, resulted in a 4-5-fold induction of D-glucose transport activity. In contrast, 24-h D-glucose starvation of primary rat brain neuronal cultures had only a marginal effect (1.5-2-fold) on D-glucose transport activity. Northern blot analysis of total cellular RNA demonstrated that under these conditions the rat brain glial cells specifically increased the steady-state level of the D-glucose transporter mRNA 4-6-fold, whereas Northern blot analysis of the neuronal cell cultures revealed no significant alteration in the amount of D-glucose transporter mRNA by D-glucose deprivation. These findings demonstrated that the D-glucose-dependent regulation of the D-glucose transporter system occurred in a brain cell type-specific manner. The ED50 for the D-glucose starvation increase in the D-glucose transporter mRNA, in the glial cell cultures, occurred at approximately 3.5 mM D-glucose with maximal effect at 0.5 mM D-glucose. Readdition of D-glucose to the starved cell cultures reversed the increase in the D-glucose transporter mRNA levels and D-glucose transport activity to control values within 24 h. The increase in the D-glucose transporter mRNA was relatively rapid with half-maximal stimulation at approximately 2 h and maximal induction by 6-12 h of D-glucose deprivation. A similar time course was also observed for the starvation-induced increase in D-glucose transport activity and D-glucose transporter protein, as determined by Western blot analysis. These results document that, in rat brain glial cells, D-glucose transport activity, protein, and mRNA are regulated by the extracellular D-glucose concentration. Further, this suggests a potential role for hyperglycemia in the down-regulation of the D-glucose transport system in vivo.     

Changes in activity of the renin-angiotensin system of the rat by induction of acute inflammation

Angiotensinogen is the precursor of biologically active peptide angiotensin II and its hepatic synthesis is increased by the induction of acute inflammation. Studies were carried out to know whether the rise in plasma angiotensinogen is actually involved in the activity of the renin-angiotensin system during acute inflammation. The plasma level of angiotensinogen in rats was increased to 2.5 times the normal level 16 h after the induction of acute inflammation by administration of lipopolysaccharide (LPS). The plasma renin concentration (PRC) was decreased to about 40% of the normal level concomitantly with a reduction of plasma renin activity (PRA) at 4 h after LPS administration. In contrast, 16 h after LPS injection, when plasma angiotensinogen showed a high level and PRC had recovered to the normal range, PRA was increased to 1.7 times the normal level. These results indicate that acute inflammation induced by LPS causes a biphasic change in the generation of angiotensin I, i.e., an early decrease depending upon the reduction of PRC and later increase depending upon elevation of the angiotensinogen concentration.     

Rapid regulation of L-type pyruvate kinase mRNA by fructose in diabetic rat liver

The effect of fructose on the induction of L-type pyruvate kinase mRNA in diabetic rat liver was studied by using a cloned cDNA probe. Fructose feeding resulted in a 5- to 6-fold increase in the L-type enzyme mRNA level after 1 to 3 days. These changes were approximately proportional to the changes in the level of translatable mRNA of this enzyme. A significant increase in total cellular L-type enzyme mRNA level was observed within 2 h after fructose feeding and the level reached a maximum after 8 h. Dietary glycerol also markedly increased the L-type mRNA level. These alterations were essentially due to the changes in the cytosolic mRNA. Northern blot analysis of total cellular RNA revealed that two L-type enzyme mRNA species with molecular sizes of 2.1 and 3.6 kilobases were proportionally increased during the fructose induction. The two mRNA forms were found in immunopurified L-type enzyme mRNA and directed synthesis of the L-type subunit in vitro; they are therefore functional mature forms. In contrast, analysis of nuclear RNA showed five putative precursor RNA species for the enzyme, up to 9.4 kilobases in length, in the liver of fructose-fed rats, while no band of the RNA species was found in the nuclei of control liver. The changes in the number of bands of these RNA species and their intensities after fructose feeding preceded the changes in the level of total cellular L-type enzyme mRNA sequences. These results indicate that dietary fructose causes a rapid increase in the level of L-type pyruvate kinase mRNA sequences by acting at the nuclear level.     

Fetal functional capabilities in response to maternal hypertonicity associated with altered central and peripheral angiotensinogen mRNA in rats

Although a number of studies have shown neural, hormonal, and behavioral capabilities in the control of body fluid regulation under conditions of dehydration in adults, limited information is available on the development of fetal functional abilities in response to osmotic challenge in rats. This study was performed to investigate the influence of maternal hypertonicity on fetal osmoregulatory capabilities at late gestational time in rats. Maternal and fetal plasma osmolality and blood sodium levels were determined and compared at continuous time points from 0.5 to 9h following maternal injection of hypertonic NaCl. Subcutaneous administration of hypertonic saline evoked a rise in plasma osmolality and sodium concentrations in maternal rats and fetuses associated with an up-regulation in angiotensinogen gene mRNA in the fetal liver and down-regulation of the same gene in the fetal brain. The increased levels of fetal blood osmolality and sodium were less than that in their mothers, and the fetus took less time to balance the enhanced osmolality and sodium concentrations. The results suggest that there may exist additional mechanisms in utero at near-term in protecting fetuses from hypertonic challenge. In addition, molecular results in the present study provide new data on fetal angiotensinogen gene expressed differently in the liver and brain under the same condition of prenatal salt loading, indicating osmotic signals of intracellular dehydration related to an acute increase in angiotensinogen mRNA in the fetal liver, and subsequent decrease in angiotensinogen mRNA levels in the fetal brain.     

Antisense oligonucleotide inhibition of angiotensinogen in the brains of rats and sheep

Phosphorothioated antisense oligodeoxynucleotides (ODNs) that were complementary to various parts of the rat or sheep mRNA encoding angiotensinogen were synthesized by conventional techniques. Their effectiveness as blockers of angiotensinogen synthesis in the brain was tested by bioassay. This involved measuring the effect of centrally administered antisense ODNs on water drinking that occurred in response to intracerebroventricular injection of hog renin. Renin-induced drinking requires brain angiotensinogen for the generation of angiotensin I and then angiotensin II to stimulate thirst. Intracerebroventricular injection of an 18-mer antisense ODN (0.5 microg twice in 24 h) complementary to the 5'-end start codon for rat angiotensinogen mRNA caused a pronounced inhibition of renin-induced drinking. This effect appeared to be specific for this region of the codon because antisense ODNs directed against other regions of rat angiotensinogen mRNA were ineffective, and renin-induced drinking was not inhibited by intracerebroventricular injection of scrambled or mismatched sequences of the effective ODN or by intraperitoneal injection of it. Intracerebroventricular injection of antisense ODN (0.5 microg twice in 24 h) did not inhibit appetite or affect water drinking in response to some other dipsogenic stimuli, thus demonstrating the specificity of its action against renin-induced drinking. By contrast, intracerebroventricular administration of 625 microg of an antisense ODN directed against the corresponding 5'-end start codon region of sheep angiotensinogen mRNA did not inhibit intracerebroventricular renin-induced drinking in sheep. These data show that while intracerebroventricularly administered antisense may be used effectively in rodents, the method is not necessarily applicable in larger mammals.     

Translation and characterization of the fatty acid synthetase messenger RNA

Fatty acid synthetase messenger RNA was obtained from rat liver polysomal RNA and then injected into Xenopus laevis oocytes. The radioactive fatty acid synthetase protein synthesized in the oocytes was identified by immunoprecipitation with anti-fatty acid synthetase antibody and the immunoprecipitate was then characterized by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel. Co-migration of authentic fatty acid synthetase and the labeled product synthesized in oocytes was observed. Based on sucrose density gradient analysis, the rat liver fatty acid synthetase mRNA has a sedimentation coefficient of approximately 33 S, which agrees with the predicted minimum size necessary to code for the fatty acid synthetase protein. In addition, this mRNA was partially purified with oligo(dT)-cellulose, which indicates that it has a polyadenylate region. The relative in vivo rate of synthesis of fatty acid synthetase and the level of fatty acid synthetase mRNA in liver were also determined during the course of dietary induction of this enzyme. The results indicate that the dietary-induced increase in the level of fatty acid synthetase is probably due to an increased level of the fatty acid synthetase mRNA.     

An analysis of the rate of metallothionein mRNA poly(A)-shortening using RNA blot hybridization.

A progressive reduction in the size of rat metallothionein-1 mRNA following induction by copper chloride or dexamethasone was demonstrated on RNA blots, and was shown to be due to shortening of the poly(A)-tail. The rate of poly(A) removal was the same in rat liver and kidney following copper chloride induction, in rat liver following dexamethasone induction, and in mouse liver following copper chloride induction. In mouse liver metallothionein-1 and 2 mRNAs were shortened at the same rate. The reduction of the poly(A) tail was more rapid in the first 5 hours (approximately 20 nucleotides/h) but much slower (approximately 3 nucleotides/h) after the poly(A)-tail had been reduced to about 60 residues. Metallothionein mRNA molecules with poly(A) tail sizes less than 30-40 nucleotides were not observed. Exonuclease digestion of the poly(A)-tail is suggested, at least in the initial rapid phase. It is hypothesized that poly(A)-tails longer than 30 are required for mRNA stability and that much longer poly(A) tails may give newly synthesized mRNA molecules a competitive advantage in protein synthesis.     

Molecular cloning of cDNA for rat and human carbamyl phosphate synthetase I

Recombinant plasmids with inserts complementary to the mRNA for carbamyl phosphate synthetase I were identified from a rat liver cDNA library by hybrid-selected mRNA translation. Four clones, the largest being 3100 base pairs, were identified for the rat liver enzyme. Using the rat liver cDNA as a probe, two homologous recombinant plasmids of approximately 1200 base pairs in length were isolated from a human liver cDNA library. Northern blot analysis of rat liver mRNA and baboon liver mRNA revealed the presence of a 5000-base mRNA homologous to both rat and human cDNA probes. No homologous mRNA was observed in mRNA from rat heart or rat kidney as is consistent with the known tissue distribution of this enzyme. The induction of carbamyl phosphate synthetase and argininosuccinate synthetase mRNA during the fetal and postnatal development of the rat was studied by dot blot analysis of isolated mRNA. The mRNA for both enzymes appeared between 17 and 19 days of fetal life and reached approximately 40% of adult levels during this period. This initial increase was followed by a rapid decline just prior to birth. The mRNA levels slowly increased during postnatal life, not reaching adult levels until after the 20th day of neonatal life. Using the human cDNA clones, the human carbamyl phosphate synthetase gene was mapped to chromosome 2 utilizing a panel of Chinese hamster X human somatic cell hybrids. Analysis of one hybrid with a human-Chinese hamster translocation provided a provisional assignment to the short arm of chromosome 2.     

Tissue-specific expression of genes encoding apolipoprotein E and apolipoprotein A-I in rabbits

We determined the site of synthesis of apolipoprotein (apo) E and apo-A-I in rabbit by measuring in vitro translational activity of their mRNAs from the liver and from the intestine. Poly(A+) RNA isolated from liver and intestinal epithelium of rabbits fed either a chow diet or a cholesterol-rich diet was translated in vitro in the rabbit reticulocyte lysate system using [35S] methionine as the labeled precursor. Newly synthesized apolipoproteins were immunoprecipitated with specific antisera and quantitated after electrophoresed on 10% polyacrylamide slab gels in the presence of 0.2% sodium dodecyl sulfate. The levels of liver apo-E and apo-A-I mRNAs from chow-fed rabbits are 0.41 and 0.002% of total translatable mRNA, respectively. The level of liver apo-A-I mRNA in the rabbit is approximately 500-fold lower than the reported level of apo-A-I mRNA in rat and human livers. Rabbit intestinal apo-E and apo-A-I mRNAs levels are 0.0036 and 0.67%, respectively. Our results indicate that in rabbits apo-E is synthesized primarily in the liver and that apo-A-I is synthesized primarily in the intestine. When rabbits are fed a cholesterol-rich diet, liver and intestinal apo-E in mRNA levels and intestinal apo-A-I mRNA levels are not changed. In contrast, the liver apo-A-I mRNA level increases 5-fold in response to the cholesterol-rich diet. However, because the intestinal liver apo-A-I mRNA level is so low, the 5-fold induction only increases liver mRNA levels to 2.7% of the corresponding intestinal apo-A-I mRNA level.     

Renin expression in the kidney and brain is reciprocally controlled by captopril

The tissue distribution of rat renin mRNA was examined. Sensitive RNase protection analyses demonstrated that renin mRNA are produced by the extra-renal tissues such as adrenal, brain, liver, lung, pituitary and testis. In response to sodium depletion and captopril treatment, the expression of mRNAs encoding rat renin were in a tissue-specific manner. The level of kidney renin mRNA remarkably increased in sodium-depleted rats treated with captopril, whereas that of brain renin mRNA definitely decreased. No significant change in the level of liver renin mRNA was observed after the same treatment. These results suggest that the expression of cerebral renin is regulated by physiological stimuli independent of its extra-cerebral expression.