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• W2081426907 abstract "Here we investigate the biochemical, molecular, and cellular changes directed toward blood pressure homeostasis that occur in the endocrine branch of the renin-angiotensin system of mice having one angiotensinogen gene inactivated. No compensatory up-regulation of the remaining normal allele occurs in the liver, the main tissue of angiotensinogen synthesis. No significant changes occur in expression of the genes coding for the angiotensin converting enzyme or the major pressor-mediating receptor for angiotensin, but plasma renin concentration in the mice having only one copy of the angiotensinogen gene is greater than twice wild-type. This increase is mediated primarily by a modest increase in the proportion of renal glomeruli producing renin in their juxtaglomerular apparatus and by four times wild-type numbers of renin-producing cells along afferent arterioles of the glomeruli rather than by up-regulating renin production in cells already committed to its synthesis. Here we investigate the biochemical, molecular, and cellular changes directed toward blood pressure homeostasis that occur in the endocrine branch of the renin-angiotensin system of mice having one angiotensinogen gene inactivated. No compensatory up-regulation of the remaining normal allele occurs in the liver, the main tissue of angiotensinogen synthesis. No significant changes occur in expression of the genes coding for the angiotensin converting enzyme or the major pressor-mediating receptor for angiotensin, but plasma renin concentration in the mice having only one copy of the angiotensinogen gene is greater than twice wild-type. This increase is mediated primarily by a modest increase in the proportion of renal glomeruli producing renin in their juxtaglomerular apparatus and by four times wild-type numbers of renin-producing cells along afferent arterioles of the glomeruli rather than by up-regulating renin production in cells already committed to its synthesis. An essential feature of complex organisms is the ability to maintain near constancy of their internal environments. Homeostasis is maintained by the operation of sophisticated systems that permit desirable physiological changes in biological variables, but that also act homeostatically if external factors cause undesirable changes in the variables. Genetic heterogeneity, such as is inherent to all outbred species including humans, also tends to cause variation in the internal environment. Yet the extent and types of homeostatic changes induced by naturally occurring genetic differences have not received much attention. We have recently been carrying out experiments aimed at identifying genes whose quantitative expression affects an important biological variable, blood pressure. To this end, we have used gene targeting in mice to alter the number of functional copies of several candidate genes, and so to produce systematic changes in their expression of the same order of magnitude as those occurring naturally in humans. Since the resulting mice are in other respects wild-type, their homeostatic systems are intact, and the mice can be used to investigate what compensations have been induced by the genetically determined differences in expression of the “titrated” genes. Because the causative genetic changes are life long, any induced compensations are categorically comparable with the lifelong adjustments that individual humans make in adjusting to the genetic heterogeneity inherent to our species. The renin-angiotensin system (RAS) 1The abbreviations used are: RAS, renin-angiotensin system; AGT, angiotensinogen; AngI, angiotensin I; AngII, angiotensin II; JGA, juxtaglomerular apparatus; ACE, Angiotensin-converting enzyme; RT-PCR, reverse transcription polymerase chain reaction; bp, base pair(s) 1The abbreviations used are: RAS, renin-angiotensin system; AGT, angiotensinogen; AngI, angiotensin I; AngII, angiotensin II; JGA, juxtaglomerular apparatus; ACE, Angiotensin-converting enzyme; RT-PCR, reverse transcription polymerase chain reaction; bp, base pair(s) is critical for controlling blood pressure and salt balance in mammals. Angiotensinogen (AGT) is the sole source of angiotensin II (AngII), the major active peptide of the system. AGT is synthesized primarily in the liver and is secreted constitutively into the blood stream. It is the substrate for renin, a highly specific protease whose only known substrate is AGT. The majority of renin synthesis and secretion into the blood stream in normal mature animals is by modified smooth muscle cells in the juxtaglomerular apparatus (JGA) of the kidney. The action of renin on AGT produces the decapeptide angiotensin I (AngI), which has no significant cardiovascular activity. Angiotensin-converting enzyme, ACE, a dipeptidase present in the blood stream as a circulating protein and in tissues as a membrane bound protein, converts AngI to the vasoactive octapeptide AngII. Genetic heterogeneity has been demonstrated at the angiotensinogen locus in humans (1Jeunemaitre X. Soubrier F. Kotelevtsev Y.V. Lifton R.P. Williams C.S. Charru A. Hunt S.C. Hopkins P.N. Williams R.R. Lalouel J.-M. Corvol P. Cell. 1992; 71: 169-180Abstract Full Text PDF PubMed Scopus (1711) Google Scholar, 2Inoue I. Nakajima T. Williams C.S. Quackenbush J. Puryear R. Powers M. Cheng T. Ludwig E.H. Sharma A.M. Hata A. Jeunemaitre X. Lalouel J.-M. J. Clin. Invest. 1997; 99: 1786-1797Crossref PubMed Scopus (508) Google Scholar), and two common alleles are associated with quantitative differences in the plasma concentration of AGT and with differences in blood pressure. In previous experiments (3Smithies O. Kim H.-S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3612-3615Crossref PubMed Scopus (162) Google Scholar, 4Kim H.-S. Krege J.H. Kluckman K.D. Hagaman J.R. Hodgin J.B. Best C.F. Jennette J.C. Coffman T.M. Maeda N. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2735-2739Crossref PubMed Scopus (580) Google Scholar) quantitative differences in expression in mice of the angiotensinogen gene (Agt) have been shown to directly cause modest changes in blood pressure. Here we explore the long term homeostatic adjustments that occur in mice attempting to restore their blood pressures to normal in the face of inheriting below normal expression of the angiotensinogen gene (Agt). Except as indicated, all mice used were F1 hybrids between the inbred strains 129 and B6 with or without a disruptive mutation in the 129-derived copy of the Agt gene. The mutation in the Agt gene was generated in embryonic stem cells from the substrain 129/OlaHsd (5Simpson E.M. Linder C.C. Sargent E.E. Davisson M.T. Mobraaten L.E. Sharp J.J. Nat. Genet. 1997; 16: 19-27Crossref PubMed Scopus (598) Google Scholar). Prior to the matings to produce the F1 hybrids, the Agt gene mutation had been maintained for several generations on the closely related substrain 129/J. The mice were fed regular chow and handled following the National Institutes of Health guidelines for the care and use of experimental animals. Blood samples were rapidly withdrawn from the descending aorta of mice after exposure to an atmosphere of CO2 (less than 1 min from loss of consciousness to the end of collection). The blood was collected into ice-cold microcentrifuge tubes containing EDTA and was immediately centrifuged to isolate plasma. Plasma AGT and (active) renin concentrations were determined by radioimmunoassay as described previously (4Kim H.-S. Krege J.H. Kluckman K.D. Hagaman J.R. Hodgin J.B. Best C.F. Jennette J.C. Coffman T.M. Maeda N. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2735-2739Crossref PubMed Scopus (580) Google Scholar). Plasma prorenin concentrations were determined after conversion to active renin by the trypsin-Sepharose 4B method (6Sealey J.E. Clin. Chem. 1991; 37: 1811-1819Crossref PubMed Scopus (196) Google Scholar). ACE activity was measured by the cleavage of a chromogenic tripeptide (7Krege J.H. John S.W.M. Langenbach L.L. Hodgin J.B. Bachman E.S. Jennette J.C. O'Brien D.A. Smithies O. Nature. 1995; 375: 146-148Crossref PubMed Scopus (602) Google Scholar), using serum isolated from blood collected retro-orbitally without anticoagulant. Blood handling and radioimmunoassays followed published methods (8Hermann K. Ganten D. Unger T. Bayer C. Lang R.E. Clin. Chem. 1988; 34: 1046-1051Crossref PubMed Scopus (49) Google Scholar, 9Kohara K. Tabuchi Y. Senanayake P. Brosnihan K.B. Ferrario C.M. Peptides (Elmsford). 1991; 12: 1135-1141Crossref PubMed Scopus (81) Google Scholar) with slight modifications. Peptides were extracted with ethanol as described in the assay procedure from the Nichols Institute (San Juan Capistrano, CA) using 600 μl of plasma pooled from three individuals matched by genotype and gender. The extracted peptide samples were divided into three equal portions and dried in a vacuum centrifuge. Single portions were used for measurement of AngI, AngII, or bradykinin. Recoveries of each peptide at completion of the extraction procedure were determined by 1125-labeled tracers to be approximately 80%. By using highly specific monoclonal antibodies for the measurements, the radioimmunoassays could be carried out without further separations. The radioimmunoassays were performed with commercially available kits for AngI (DuPont), AngII (Nichols), and bradykinin (Peninsula, Belmont, CA). Tissues were rapidly dissected after withdrawing blood. Total RNA was isolated conventionally (10Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63088) Google Scholar) using the TRI REAGENTTM procedure (Molecular Research Inc., Cincinnati, OH). DNA fragments from theAgt, renin, and β-actin genes were prepared by PCR using strain 129/OlaHsd mouse genomic DNA as the template and the following probes designed from published sequences: a 390-bp exon 2 fragment from the Agt gene (11Clouston W.M. Evans B.A. Haralambidis J. Richards R.I. Genomics. 1988; 2: 240-248Crossref PubMed Scopus (46) Google Scholar); a 290-bp exon 9 fragment from the mouse renin gene Ren-1 d (12Burt D.W. Mullins L.J. George H. Smith G. Brooks J. Pioli D. Brammar W.J. Gene (Amst.). 1989; 84: 91-104Crossref PubMed Scopus (27) Google Scholar); and a 250-bp fragment from the mouse β-actin gene (13Alonso S. Minty A. Bourlet Y. Buckingham M. J. Mol. Evol. 1986; 23: 11-22Crossref PubMed Scopus (604) Google Scholar). A 418-bp fragment corresponding to nucleotides 2523–2931 of mouse ACE cDNA (14Bernstein K.E. Martin B.M. Edwards A.S. Bernstein E.A. J. Biol. Chem. 1989; 264: 11945-11951Abstract Full Text PDF PubMed Google Scholar) was cloned after RT-PCR using total RNA from the lung as template. All of the fragments were subcloned into a Bluescript(KS) vector for in vitrotranscription (15Melton D.A. Krieg P. Rebagliati M.R. Maniatis T. Zinh K. Green M.R. Nucleic Acids Res. 1984; 12: 7035-7055Crossref PubMed Scopus (4054) Google Scholar). 32P-Labeled antisense riboprobes were synthesized by the manufacturer's protocols using a MAXI scriptTM transcription kit (Ambion Inc., Austin, TX). Unlabeled sense RNAs were prepared with the same transcription system. The sense RNAs were gel-purified and stored at −70 °C. The procedure described by Azrolan and Breslow (16Azrolan N. Breslow J.L. J. Lipid Res. 1990; 31: 1141-1146Abstract Full Text PDF PubMed Google Scholar) with minor modifications was used for RNase protection assay. All reactions were carried out in duplicate for each sample in all experimental groups. A standard curve was generated using sense RNAs. Primer preparation and primer extension analysis for the mouse renin genes were slightly modified versions of published procedures (17Field L.J. Gross K.W. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6196-6200Crossref PubMed Scopus (68) Google Scholar, 18Fabian J.R. Field L.J. McGowan R.A. Mullins J.J. Sigmund C.D. Gross K.W. J. Biol. Chem. 1989; 264: 17589-17594Abstract Full Text PDF PubMed Google Scholar). The primer was a 38-mer oligonucleotide complementary to Ren-1 C mRNA from positions 1039 through 1076 of the cDNA sequence (19Kim W.S. Murakami K. Nakayama K. Nucleic Acids Res. 1989; 17: 9480PubMed Google Scholar). We determined that the nucleotide sequence of this region is identical in the strain B6 Ren-1 c gene and the strain 129Ren-1 d and Ren-2 genes. Autoradiographic bands were quantitated with an NIH image computer program (version 1.55). Quantitative RT-PCR (20Paul M. Wagner J. Dzau V.J. J. Clin. Invest. 1993; 91: 2058-2064Crossref PubMed Scopus (219) Google Scholar) was used to assess expression of the gene coding for the type 1A receptor for AngII. Total RNA from the kidney was reversely transcribed to cDNA (21Wang A.M. Doyle M.W. Mark D.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9717-9721Crossref PubMed Scopus (1615) Google Scholar). A template plasmid for preparing the internal standard was constructed by making a 120-bp deletion (PvuII/DraI sites) in aPlmI/SacI fragment from the mouse type 1A receptor gene. PCR primers specific for the type 1A gene were designed from sequences of the type 1A and 1B genes (22Sasamura H. Hein L. Krieger J.E. Pratt R.E. Kobilka B.K. Dzau V.A. Biochem. Biophys. Res. Commun. 1992; 185: 253-259Crossref PubMed Scopus (281) Google Scholar); they are 5′-ACGAGTCCCGGAATTACACG-3′ for the sense primer and 5′-GCGTGCTCATTTTCGTAGACAGG-3′ for the antisense primer. Competitive PCR was performed in the presence of an internal standard, yielding a 320-bp fragment. The RT product corresponding to the type 1A mRNA is 440 bp long. The amount of the full-length product relative to the internal standard was determined after hybridization to labeled full-length fragment as a probe. Immunohistochemical detection of renin was as described previously (23Gomez R.A. Lynch K.R. Chevalier R.L. Everett A.D. Johns D.W. Wilfong N. Peach M.J. Carey R.M. Am. J. Physiol. 1988; 254: F900-F906PubMed Google Scholar, 24Romano L.A. Ferder L. Inserra F. Ercole L. Gomez R.A. Hypertension (Dallas). 1994; 23: 889-893Crossref PubMed Google Scholar). Briefly, after deparaffinization, 7-μm kidney sections were incubated with a polyclonal renin antibody (1:10,000, gift from Dr. Tadashi Inagami, Vanderbilt University, Nashville, TN). The high specificity and characterization of this renin antibody has been documented previously (25Naruse K. Takii Y. Inagami T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7579-7583Crossref PubMed Scopus (82) Google Scholar). Immunocytochemistry was done with kidneys from the Agt one-copy and wild-type mice. Two to four sections per kidney were examined by direct microscopic visualization. The total number of glomeruli, the number with renin-positive JGA, and the number with renin-positive cells upstream of the JGA were counted in each section. The percentage of renin-positive JGA was determined as (the number of renin-positive JGA in all sections) × 100/(the total number of glomeruli observed). The number of cells positive for renin, including those in the JGA itself, along the afferent arterioles of glomeruli having upstream renin-positive cells was also counted in each section. The figures thus obtained from each slide were averaged for each animal. To determine the area of juxtaglomerular apparatuses in theAgt one-copy and wild-type mice, 10 random fields of each section were captured with a video camera. Every section was screened using the same magnification (× 400), and only the JGA with a classic donut-shaped outline were evaluated. All the images were studied with an image analysis software (MochaTM, version 1.02, Jandel Scientific). Using the manual measurement mode, the perimeter of each JGA was outlined, and its area was determined by summing the number of pixels contained within the outline. To determine the number of cells in the JGA that had been evaluated for area, the number of nuclei observed within the outlined perimeter was counted. To obtain an integrated view of the distribution of renin within the kidney, the entire renal arterial tree was dissected as described previously in rats and mice (26Casellas D. Dupont M. Kaskel F.J. Inagami T. Moore L.C. Am. J. Physiol. 1993; 265: F151-F156PubMed Google Scholar, 27Hilgers K.F. Reddi V. Krege J.H. Smithies O. Gomez R.A. Hypertension (Dallas). 1997; 29: 216-221Crossref PubMed Google Scholar) and stained for renin. The distribution of renin within the kidney was classified as described previously (28Reddi V. Zaglul A. Pentz E.S. Gomez R.A. J. Am. Soc. Nephrol. 1998; 9: 63-71Crossref PubMed Google Scholar). In a type I distribution, renin is present along the whole length of the afferent vessel. In type II, renin extends upstream from the glomerulus but does not occupy the whole length of the vessel. In type III, renin is present as rings along the afferent vessel. In type IV, renin is restricted to the classical juxtaglomerular localization. In type V, no renin is found in the arteriole. All values are expressed as mean ± S.E. The two-tailed t test was used for statistical evaluations. The experimental animals used for investigating long term homeostatic compensations in the RAS have a single functional copy of the Agt gene and one disrupted by gene targeting. We refer to them as Agt one-copy mice, and their blood pressures are about 8 mmHg (approximately 7%) below the pressures of wild-type mice with two copies of the gene (4Kim H.-S. Krege J.H. Kluckman K.D. Hagaman J.R. Hodgin J.B. Best C.F. Jennette J.C. Coffman T.M. Maeda N. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2735-2739Crossref PubMed Scopus (580) Google Scholar). Except when indicated, the mice studied were F1 progeny-derived from the two inbred strains 129 and C57BL/6 (B6) and so were genetically identical except for having different numbers of functional Agt genes. Because of this genetic uniformity, even small differences between the mice can be ascribed directly to the difference in Agt gene copy number. To determine what components are homeostatically adjusted in the Agtone-copy animals, the steady state concentrations of the major RAS protein components present in plasma were compared in theAgt one-copy animals and their wild-type controls. The resulting data, Table I and Fig.1 (below), show two major differences. First, the plasma AGT concentration in the Agt one-copy mice is markedly reduced, to 33–37% of the AGT concentration in the controls (p < 0.01), which is also significantly less than the 50% expected if the amount of protein were directly related to gene copy number (p < 0.01 versus 50%). A possible complication affecting this observation is that the functional Agt gene in the one-copy animals is derived from mouse strain B6, whereas the wild-type two-copy animals have one copy from strain B6 and one from strain 129. However, a comparison of AGT levels in wild-type inbred strain B6 and 129 mice shows that B6 mice have higher AGT levels (558 ± 30 AngI ng/ml/h, in six females) than do 129 mice (426 ± 11 AngI ng/ml/h, in six females), so that if strain differences in Agt gene expression persist in the F1 hybrids, the AGT concentration in theAgt one-copy animals should be even more than 50% of the wild-type F1 animals. We conclude that the plasma concentration of AGT shows no evidence of any compensatory increase in the Agtone-copy animals.Table IPlasma proteins and peptides in F1 Agt wild-type and F1 Agt one-copy miceMalesFemalesnAgtwtAgt one-copyAgt wtAgtone-copyAGT6498 ± 23164 ± 6 (33)ap < 0.01 versus wild-type.550 ± 14201 ± 16 (37)ap < 0.01 versus wild-type.Renin638 ± 185 ± 6 (222)bp < 0.001 versus wild-type.50 ± 4131 ± 12 (262)bp < 0.001 versus wild-type.Prorenin6244 ± 3572 ± 51 (234)ap < 0.01 versus wild-type.NDNDACE3400 ± 29360 ± 15 (90)cp = 0.15 versus wild-type.246 ± 15251 ± 11 (102)cp = 0.15 versus wild-type.AngI359 ± 334 ± 4 (58)bp < 0.001 versus wild-type.60 ± 245 ± 3 (75)bp < 0.001 versus wild-type.AngII323 ± 512 ± 1 (50)ap < 0.01 versus wild-type.18 ± 111 ± 2 (62)ap < 0.01 versus wild-type.Bradykinin394 ± 496 ± 2 (103)dp = 0.87 versus wild-type.70 ± 264 ± 1 (93)dp = 0.87 versus wild-type.AGT, renin, and prorenin: ng of AngI/ml/h. ACE activity: units/liter. AngI, AngII, and bradykinin: pg/ml. Values are means ± S.E. Values in parentheses are percent relative to wild-type. n, number of animals; ND, not determined; wt, wild type.a p < 0.01 versus wild-type.b p < 0.001 versus wild-type.c p = 0.15 versus wild-type.d p = 0.87 versus wild-type. Open table in a new tab AGT, renin, and prorenin: ng of AngI/ml/h. ACE activity: units/liter. AngI, AngII, and bradykinin: pg/ml. Values are means ± S.E. Values in parentheses are percent relative to wild-type. n, number of animals; ND, not determined; wt, wild type. The second major difference is that, in marked contrast to the absence of any detectable compensation in AGT plasma concentration, the plasma renin concentration in the Agt one-copy animals is very significantly higher (240%) than in the wild-type controls (p < 0.001), indicating a marked homeostatic adjustment in this component of the RAS. In mice and humans, renin is secreted into the circulation by renin-producing cells partly as enzymatically inactive prorenin and partly as enzymatically active renin (29Sealey J.E. Atlas S.A. Laragh J.H. Endocr. Rev. 1980; 1: 365-391Crossref PubMed Scopus (210) Google Scholar). The observed homeostatic increase in plasma renin could therefore be partly or wholly the consequence of a change in the proportion of the secreted protein in the active versus inactive form of renin. To investigate this possibility, we determined the plasma concentration of prorenin inAgt one-copy and in wild-type animals as well as the concentration of (active) renin. The plasma prorenin concentration in the Agt one-copy male animals was significantly higher (234%; p < 0.01) than in the wild-type controls (Table I). This increase in plasma prorenin is virtually identical to that of the plasma active renin, so that the same ratio of prorenin and active renin is observed in Agt one-copy and wild-type mice. We conclude that a change in the ratio of these two products is not part of the homeostatic adjustment made in the Agt one-copy mice. To determine the net effect of the observed increase in plasma renin concentration combined with the observed decrease in plasma AGT concentration, we compared the steady state concentrations of AngI inAgt one-copy and wild-type mice. The results show that theAgt one-copy animals have AngI levels that are 58% and 75% of wild-type in males and females, respectively. Thus the combined effect of the two changes is a partial but not complete restoration of the AngI concentrations to the wild-type level (p < 0.001 for one-copy versus wild-type), albeit at the expense of decreasing the steady state concentration of AGT below 50% of wild-type. An additional possible means of compensating for the less than normal AGT and AngI plasma levels in the one-copy animals would be via a homeostatically induced increase in the level of the converting enzyme ACE. Measurements of serum ACE activities (Table I), however, show no significant differences (p = 0.15) between theAgt one-copy mice and the wild-type controls. An additional indicator of possible changes in ACE function is the plasma bradykinin concentration, since this octapeptide is destroyed by the enzyme. We found that the bradykinin levels were not different between theAgt one-copy mice and the wild-type mice (p= 0.87). Thus we conclude that homeostatic compensation has not been induced at the level of the converting enzyme or of the bradykinin peptide. The major effector peptide of the RAS is the octapeptide AngII. A measure of changes in the net status of the circulating arm of the system can therefore be obtained by comparing the steady state plasma concentrations of AngII in Agt one-copy and wild-type mice. The resulting data show that plasma AngII in the Agtone-copy males and females are, respectively, 50% and 62% of the levels in the wild-type animals. These levels are significantly less than wild-type (p < 0.01), indicating that homeostasis is incomplete, as is reflected by the residual differences in blood pressure between Agt one-copy and wild-type animals. In summary, (Fig. 1), measurements of the expression of the protein and peptide components of the endocrine RAS show clear evidence that a major homeostatic compensation occurs in plasma renin concentrations in response to a genetic reduction in Agt gene expression. Other components of the system either show no changes or have changes that appear to be passive and secondary to the genetic reduction in AGT levels and the consequent homeostatic increase in renin. The final result is a steady state concentration of AngII in Agtone-copy animals that is still significantly less than normal. To ascertain whether the changes seen in the circulating protein components of the RAS are present at the level of transcriptional products, we used an RNase protection assay to determine the amounts of the relevant mRNAs in tissues that make the largest contribution to the plasma inAgt one-copy and wild-type animals. The major site of synthesis of AGT secreted into blood is the liver (30Nasjletti A. Masson G.M.C. Can. J. Physiol. Pharmacol. 1971; 49: 931-932Crossref PubMed Scopus (34) Google Scholar), which also contains the highest abundance of AGT mRNA. Fig.2A presents the data for AGT mRNA inAgt one-copy and wild-type F1 males and females and in strain B6 and 129 inbred wild-type males. More mRNA is present in the female mice than in the males (a disparity also observed in the plasma AGT). But regardless of gender the liver AGT mRNA levels inAgt one-copy animals are clearly reduced compared with those in the wild-type animals (54% in males; 53% in females;p < 0.001). Recollecting that the single functionalAgt gene in the one-copy animals is derived from strain B6 and noting from Fig. 2 A that liver AGT mRNA levels in strain B6 are 1.2 times the corresponding levels in strain 129, we conclude that the liver AGT mRNA data agree with the plasma protein data in indicating no homeostatic compensation in the Agtone-copy mice in the transcription of the remaining functionalAgt gene in the primary tissue of AGT synthesis. The high steady state plasma renin (and prorenin) concentrations observed in Agt one-copy animals suggest a substantially increased level of renin gene transcription. Fig. 2 Bpresents the relevant data and shows that the steady state renin mRNA contents of the kidneys of the Agt one-copy males and females are respectively 182 and 165% of the wild-type two-copy controls (p < 0.001). Thus a major part of the homeostatic adjustment in renin production is a consequence of an increase in amount of renin mRNA. Since the steady state serum ACE activities of the experimental and control mice do not differ significantly, we expected to see no differences in the ACE mRNA levels in tissues in which ACE is synthesized. The lungs are a major site of ACE synthesis in both sexes (31Ng K.K.F. Vane J.R. Nature. 1967; 275: 762-766Crossref Scopus (348) Google Scholar). In addition, a truncated form of ACE is synthesized in the testis from a testis-specific promoter (32Howard T. Balogh R. Overbeek P. Bernstein K.E. Mol. Cell. Biol. 1993; 13: 18-27Crossref PubMed Scopus (101) Google Scholar). Fig. 2 C presents data showing that the ACE mRNA contents of the lungs of Agtone-copy males and females are slightly increased (108 and 105%) relative to their two-copy controls, but the difference is not statistically significant (p = 0.09). The ACE mRNA level in the testis was also slightly higher (data not shown), but again the difference was not significant (108% wild-type,p = 0.65). Thus there is no evidence for significant homeostatic compensation at the level of ACE mRNA. In the mouse, three receptors (types 1A, 1B, and 2) control the cellular and physiologic actions of AngII (22Sasamura H. Hein L. Krieger J.E. Pratt R.E. Kobilka B.K. Dzau V.A. Biochem. Biophys. Res. Commun. 1992; 185: 253-259Crossref PubMed Scopus (281) Google Scholar, 33Whitebread S. Mele M. Kamber B. deGasparo M. Biochem. Biophys. Res. Commun. 1989; 163: 284-291Crossref PubMed Scopus (758) Google Scholar). The results of administering receptor antagonists that specifically block the actions of either the type 1 or the type 2 receptors establish that blood pressure changes are chiefly executed by the type 1 receptors (34Timmermans P.B.M.W.M. Wong P.C. Chiu A.T. Herblin W.F. Benfield P. Carini D.J. Lee R.J. Wexler R.R. Saye J.A.M. Smith R.D. Pharmacol. Rev. 1993; 45: 205-251PubMed Google Scholar). Genetic experiments disrupting the genes coding for the type 1A receptor gene (35Ito M. Oliverio M.I. Mannon P.J. Best C.F. Maeda N. Smithies O. Coffman T.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3521-3525Crossref PubMed Scopus (545) Google Scholar, 36Oliverio M.I. Best C.F. Kim H.S. Arendshorst W.I. Smithies O. Coffman T.M. Am. J. Physiol. 1997; 272: F515-F520PubMed Google Scholar) or the type 1B receptor gene (37Chen X. Li W. Yoshida H. Tsuchida S. Nishimura H. Takemoto F. Okubo S. Foqo A. Matsusaka T. Ichikawa I. Am. J. Physiol. 1997; 272: F299-F304Crossref PubMed Google Scholar, 53Oliverio M.I. Kim H.-S. Ito M. Le T. Audoly L. Best C.F. Hiller S. Kluckman K. Maeda N. Smithies O. Coffman T.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15496-15501Crossref PubMed Scopus (278) Google Scholar) show that approximately 90% of the endocrine pressor effects of AngII are via by the type 1A receptor. Another possible means of homeostatic adjustments in the face of a chronic decrease in blood pressure would therefore be to increase expression of the type 1A receptor. However, comparison of the type 1A receptor gene expression by RT-PCR (Fig.3) in Agt wild-type mice (lane 1) and the Agt one-copy mice (lane 2) revealed no detectable differences, although the same assay" @default.
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• W2081426907 title "Homeostasis in Mice with Genetically Decreased Angiotensinogen Is Primarily by an Increased Number of Renin-producing Cells" @default.
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