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The primary structures of the angiotensinogen precursor and the low molecular weight (LMW) kininogen precursors have been deduced by determining the nucleotide sequences of cloned DNAs complementary to their mRNAs. The angiotensinogen precursor consists of a mature angiotensinogen of 453 amino acid residues and a putative signal peptide of 24 amino acid residues. An angiotensin moiety is located at the amino-terminal part of angiotensinogen, preceded directly by the signal peptide and followed by a large carboxyl-terminal sequence that contains two internally homologous sequences and three potential glycosylation sites. The LMW kininogen precursors are encoded by two very similar but distinct mRNAs and composed of 436 and 434 amino acid residues. Both kininogens contain two internally homologous sequences in which all amino acid differences between the two kininogens are located. This suggests that these homologous regions may be biologically significant in relation to the existence of two LMW kininogens.

A mixture of tetradecamer oligodeoxyribonucleotides complementary to the codons specifying the carboxyl-terminal sequence, Ile-His-Pro-Phe-His, of angiotensin was chemically synthesized as two pools and used for the isolation of a cDNA clone specific for angiotensinogen from a cDNA bank of rat liver mRNA sequences. The two pools (oligo 1 and oligo 2), each containing 24 oligodeoxyribonucleotides, were first used as primers to initiate reverse transcription of rat liver mRNA. One of the pools (oligo 1) was found to prime a specific 32P-labeled cDNA of approximately 160 nucleotides that contained the anticoding sequence corresponding exactly to the amino acid sequence of rat angiotensin. This cDNA, in turn, was used to rescreen cDNA clones that were isolated by initially selecting the rat liver cDNA bank by hybridization with the oligo 1 mixture. One clone thus obtained, designated pRag16, was subjected to nucleotide sequence analysis and verified to contain a nearly full-length cDNA sequence coding for rat angiotensinogen precursor. The deduced amino acid sequence indicates that the precursor molecular consists of angiotensinogen of 453 amino acid residues and a putative signal peptide of 24 amino acid residues. The predicted molecular weight and amino acid composition of angiotensinogen agree well with those determined by using the purified protein. An angiotensin moiety is located at the amino-terminal part of angiotensinogen, preceded directly by the signal peptide and followed by a large carboxyl-terminal sequence that contains two internally homologous sequences and three potential glycosylation sites.

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin. Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be diferent from the amino acid sequence previously published elsewhere. S 1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.

The human renin gene was isolated from a Charon 4A human genomic library and characterized. The gene spans about 11.7 kilobases and consists of 10 exons and 9 introns that map at points that could be variable surface loops of the enzyme. The complete coding regions, the 5'- and 3'-flanking regions, and the exon-intron boundaries were sequenced. The active site aspartyl residues Asp-38 and Asp-226 are encoded by the third and eighth exons, respectively. The extra three amino acids (Asp-165, Ser-166, Glu-167) that are not present in mouse renin are encoded by the separate sixth exon, an exon as small as 9 nucleotides. The positions of the introns are in remarkable agreement with those in the human pepsin gene, supporting the view that the genes coding for aspartyl proteinases have arisen as the result of duplication of a common ancestral gene. As in most eukaryotic genes, the putative T-A-T-A and C-A-A-T sequences, which may play a role in the initiation of gene transcription, are found in the vicinity of -29 and -51 nucleotides of the cap site. Further upstream, at nucleotides -456 to -451, is located the hexanucleotide T-G-T-T-C-T, which has recently been suggested as a binding site for the glucocorticoid receptor. In the 3'-flanking region, there is the conserved hexanucleotide sequence A-A-T-A-A-A, thought to be necessary for polyadenylylation. Blot-hybridization analyses of the isolated gene clone and the total cellular DNA after digestion with restriction enzymes revealed that human renin is encoded by a single gene.

The expression of messenger (m)RNA for renin, angiotensinogen and atrial natriuretic factor (ANF) was investigated in rats on different sodium intakes. Messenger RNA was measured by a radiodensitometric hybridization assay. In the high-sodium state, renal renin mRNA decreased, but it increased in the low-sodium state. A further increase in renin mRNA was seen in the low-sodium state after captopril administration. Angiotensinogen mRNA levels in the liver, kidney and brain were altered by varying sodium intake. In the high-sodium state angiotensinogen mRNA decreased, but in the low-sodium state it increased. After treatment with captopril, angiotensinogen mRNA levels decreased in the liver and kidney. Angiotensinogen mRNA showed tissue specificity for expression, especially in the brain. Atrial ANF mRNA levels changed slightly with different levels of sodium intake.

The mammalian cardiac atrium has recently been shown to contain numerous peptides that exert marked effects on kidney function and vascular resistance1–9. With one exception, the peptides have potent natriuretic and diuretic activities, and sequence similarities suggest that they may be derived from a common atrial natriuretic factor (ANF) precursor10–14. The exception, cardiodilatin (CDD), differs from the ANF peptides in possessing potent vasorelaxant activity, but not natriuretic and diuretic activities9. Here we report the cloning and sequence analysis of the cDNA for the human precursor protein (preproCDD–ANF) containing both the CDD and ANF sequences. The CDD sequence represents the N-terminal sequence preceded directly by a signal peptide, while the ANF sequence is present at the C-terminal end of the protein. Using hybridization analysis, we further show that the amount of rat preproCDD–ANF mRNA, which is synthesized selectively in the atria but not the ventricles, markedly decreases on water deprivation, suggesting that the water–electrolyte balance may be an important factor in the regulation of the expression of the preproCDD–ANF gene.

In order to further elucidate the expression of the atrial natriuretic factor (ANF) gene in spontaneously hypertensive rats (SHR) and the substrain, stroke-prone SHR (SHRSP), ANF messenger (m)RNA levels in the atrium and in the ventricle were measured in neonates of SHR and SHRSP and were compared with those in control Wistar-Kyoto rats (WKY). The levels of ANF mRNA in ventricles of the three strains reached approximately 7% of those in atria; however, no significant difference was observed in atrial and ventricular ANF mRNA levels among the three strains. These results demonstrate that the expression of the ANF gene in hearts from neonates of WKY, SHR and SHRSP is similar among the three strains.

Complementary DNA clones, pHEW1 and pRE2, coding for hamster and rat polypeptide chain elongation factor 2 (EF-2), respectively, were isolated and sequenced. It was shown that the cDNA insert in pHEW1 contains a 2574-base-pair open reading frame coding for an 857-amino acid polypeptide with Mr 95,192, excluding the initiation methionine. Comparative studies of sequence homology among EF-2 and several GTP-binding proteins show that five regions in the amino-terminal position of EF-2, corresponding to about 160 amino acids, show homology with GTP-binding proteins, including protein synthesis elongation and initiation factors, mammalian ras proteins, and transducin. The carboxyl-terminal half of EF-2 contains several regions that have 34-75% homology with bacterial elongation factor G. These results suggest that the amino-terminal region of EF-2 participates in the GTP-binding and GTPase activity whereas the carboxyl-terminal region interacts with ribosomes. Finally, the sequence provides direct evidence that diphthamide (2-[3-carboxy-amido-3-(trimethylammonio)propyl]histidine), the site of ADP-ribosylation by diphtheria toxin, is produced by post-translational modification of a histidine residue in the primary translational product.

cDNA clones for the (S)-tetrahydroberberine (H4Ber) oxidase of cultured berberine-producing Coptis japonica cells were isolated by screening a C. japonica cDNA library with synthetic nucleotides that can encode the NH2-terminal sequence of this enzyme. Analyses of the nucleotide sequences of the cloned cDNA inserts revealed a 759-base-pair open reading frame that encoded a 253-amino acid polypeptide with a Mr of 27,089 and NH2-terminal and internal sequences identical with those of the (S)-H4Ber oxidase, as determined by microsequencing methods. Escherichia coli were transformed with an expression vector carrying (S)-H4Ber oxidase cDNA. The transformed bacteria were induced to overproduce a 28-kDa protein that reacted with Coptis (S)-H4Ber oxidase-specific antibody. A comparison of the derived amino acid sequence of (S)-H4Ber oxidase with sequences in the protein data base of the Protein Research Foundation showed a marked similarity between (S)-H4Ber oxidase and the NH2-terminal portion of mouse P1-450, which is encoded by a single exon of the mouse P1-450 gene. The availability of cloned cDNA for (S)-H4Ber oxidase allows use of the methods of molecular biology to study the regulation of (S)-H4Ber oxidase gene expression in cultured C. japonica cells in relation to berberine biosynthesis.

In order to investigate the structures and expressions of the angiotensinogen and kininogen genes, we have isolated cDNA and genomic clones and characterized their structures. The following results were obtained. (a) The primary structures of rat and human angiotensinogens and bovine, human, and rat kininogens were determined, and they exhibit characteristic features. (b) Angiotensinogen and alpha 1-antitrypsin are related in not only protein structures but also gene organizations. (c) Angiotensinogen mRNA is synthesized not only in the liver but also in the brain, kidney, lung, adrenal gland, and ovary. (d) Angiotensinogen mRNA markedly increased in the liver but not in the brain after induction of acute inflammation. (e) Low molecular weight (LMW) and high molecular weight (HMW) kininogen mRNAs are generated from a single gene by alternative RNA processing events. (f) At least four types of kininogen mRNAs exist in rat liver. Two of them are LMW and HMW kininogen mRNAs, while the other two encode additional forms of kininogens (T-kininogens). (g) Two T-kininogen mRNAs but not LMW and HMW kininogen mRNAs dramatically increased after induction of acute inflammation, and T-kininogen turned out to be identical to alpha 1-major acute phase protein. (h) Kininogens have multifunctional domains; an amino-terminal domain for cystein proteinase inhibitor, a bradykinin moiety, and in the case of HMW kininogen a carboxyl-terminal domain for a cofactor of blood coagulation.

We investigated the influences of captopril (CAP) and hydralazine (HYD) on the ANP mRNA level in the hypertrophied left ventricle (LV) of spontaneously hypertensive rats (SHR). Male SHRs (16 weeks of age) were given CAP (35 mg/kg/day) or HYD (3.5 mg/kg/day) for two weeks. Both drugs reduced blood pressure by a similar magnitude. Treatment with CAP caused a reduction in the ANP mRNA level in LV by 62%, and a reduction in the weight of the LV. The ANP mRNA level in LV of the HYD-treated rats was also decreased, but only by 31%. HYD did not affect LV hypertrophy. ANP gene expression in LV of SHR might be effectively suppressed by a reduction of blood pressure and also by the concomitant attenuation of hypertrophy.

Summary Brain natriuretic peptide (BNP) is a new type of natriuretic peptide recently identified in porcine brain. Since the highest concentration of BNP was found in the cardiac atrium, the cDNA library of porcine cardiac atrium was constructed, and the cDNA clone encoding a BNP precursor was isolated and sequenced. The precursor for porcine BNP (porcine prepro-BNP) is 131 amino acids in length, including a 25 residue putative signal peptide at the N-terminus. Porcine BNP structure is located at the C-terminus of the precursor and is directly followed by a termination codon. Based on structural data recently obtained for γ-BNP (a main storage form of BNP in the heart), prepro-BNP is processed to 106-residue γ-BNP by removal of the signal peptide in the heart, and to low molecular weight forms, such as BNP-26 and BNP-32, in the brain.

We studied the methylation pattern of the alpha 2 (type 1) collagen gene in DNA from five cell types with varying rates of type I collagen synthesis: chick embryo fibroblast (CEF), CEF transformed by Rous sarcoma virus, erythrocyte, brain and sperm. The methylation-sensitive restriction enzymes, Msp I, Hpa II, Ava I and Sma I were used to detect methylation in three regions of the alpha 2 (type I) collagen gene: a 5.7 kb region, which includes the start site of transcription and the first two exons of the collagen gene; a 5.2 kb region containing exons in the middle of the gene; and a 3.5 kb region containing exons in the 3' portion of the gene. The DNA around the start site of transcription is not methylated whether or not the cells synthesize collagen. In contrast, the DNA from the central and 3' region of the gene is methylated to about the same extent whether or not the cells make collagen. Our data indicate that a gene that is methylated can be actively transcribed and that the level of expression of the alpha 2 (type I) collagen gene seems to be independent of methylation.

Cloned cDNA sequences for human preangiotensinogen have been isolated from a human liver cDNA library by hybridization with a restriction fragment derived from a previously cloned cDNA for rat preangiotensinogen. Analyses by nucleotide sequence determination, S1 nuclease mapping, and RNA blot hybridization indicate that human preangiotensinogen is encoded by two mRNAs that differ only in the length of the 3'-untranslated region. The deduced amino acid sequence shows that the mature angiotensinogen consists of 452 amino acid residues with the angiotensin sequence at its amino-terminal portion. Two potential initiation sites have been discussed. These are the methionine codon located at the position exactly corresponding to the initiation site of rat preangiotensinogen mRNA and an additional methionine codon positioned nearest the 5' end of the mRNA. The amino acid sequences starting at either of the initiation sites and preceding the angiotensin sequence constitute a large number of hydrophobic amino acid residues, thus representing the signal peptide characteristic of the secretory proteins. Human and rat preangiotensinogens show that 63.6% of the amino acid positions of the two proteins are identical. However, the amino-terminal portions directly distal to angiotensin I diverge markedly between the two proteins and differ in their possible glycosylation sites. These structural differences may contribute to the known species specificity exhibited by renin.

We measured the levels of messenger RNA of the human multidrug-resistance gene (MDR1) in 25 urogenital tumors before chemotherapy. Many of the renal cell carcinomas continued to express MDR1 gene at high levels, reflecting the increased expression of MDR1 RNA in normal kidneys. In other urogenital tumors, the MDR1 RNA levels were low reflecting low MDR1 RNA levels in normal bladder, prostate and testis. For comparative purposes, we performed in vitro chemosensitivity testing on many tumor samples using soft agar culture techniques. Vinblastine sensitivity in vitro inversely correlated with MDR1 RNA levels (p less than 0.01). Moreover, mean sensitivity of seven renal cell carcinomas to vinblastin was significantly lower than that of the other seven cancers (p less than 0.05). As for doxorubicin, mean sensitivity of six renal cell carcinomas was lower than the others (p less than 0.1). These results suggest that the high MDR1 RNA levels in renal cell carcinomas are associated with intrinsic multidrug-resistance.

A rat genomic DNA segment containing the angiotensinogen gene has been isolated from a gene library by hybridization with a restriction fragment derived from a previously cloned cDNA for rat angiotensinogen. Restriction mapping and nucleotide sequence analysis of the cloned DNA fragments have indicated that the rat angiotensinogen gene is approximately 11.8 kilobase pairs long and consists of five exons separated by four introns. Because it has recently been reported that the sequence of angiotensinogen significantly resembles those of alpha 1-antitrypsin and ovalbumin and also that the exon-intron arrangements of the alpha 1-antitrypsin and ovalbumin genes are completely different, this study compares the structural organization of the angiotensinogen gene with those of the alpha 1-antitrypsin gene and the ovalbumin gene. The comparison has revealed that the four introns of the angiotensinogen gene are all located at the positions equivalent to or corresponding to those observed in the alpha 1-antitrypsin gene, suggesting that the angiotensinogen gene and the alpha 1-antitrypsin gene have diverged from a common ancestor gene. This finding raises interesting possibilities regarding the biological function of angiotensinogen and the evolution of the angiotensinogen gene.

Brain natriuretic peptide (BNP) is a new type of natriuretic peptide recently identified in porcine brain. Since the highest concentration of BNP was found in the cardiac atrium, the cDNA library of porcine cardiac atrium was constructed, and the cDNA clone encoding a BNP precursor was isolated and sequenced. The precursor for porcine BNP (porcine prepro-BNP) is 131 amino acids in length, including a 25 residue putative signal peptide at the N-terminus. Porcine BNP structure is located at the C-terminus of the precursor and is directly followed by a termination codon. Based on structural data recently obtained for gamma-BNP (a main storage form of BNP in the heart), prepro-BNP is processed to 106-residue gamma-BNP by removal of the signal peptide in the heart, and to low molecular weight forms, such as BNP-26 and BNP-32, in the brain.

We have determined the size and sequence of 8 exons in the gene that specifies chick type I α2 collagen. These 8 exons represent three different segments of the gene, but all encode information for the helical portion of the protein. Seven exons have a length of 54 bp, and the 8th has a size of 99 bp. The sequences within these exons vary except for the glycine codons, which occur every third triplet. Each exon begins with a glycine codon and ends with a triplet that precedes a glycine codon. The size and the sequences of the introns do not show any homology except at their ends. Of 7 introns examined the first six bases at the 5′ end of 5 of these are identical. The sequences at the 3′ end of the introns also show homologies. Our results imply that the ancestral gene for collagen arose by multiple duplications of a single genetic unit containing a 54 bp coding segment. The sequences within these exons drifted by successive point mutations and in some cases by additions or deletions of 9 bp or multiples of 9 bp.

Two types of rat kininogen mRNAs exhibit marked differences in the regulation of their expressions. The two T kininogen (T) mRNAs considerably increase under acute inflammation conditions, although no such increase occurs in the high molecular weight and low molecular weight K kininogen (K) mRNAs encoded by the same gene. This investigation examines the sequences of the 5' portions of the K and the two T genes and analyzes transcription initiation sites and their utilization at differently regulated conditions of the K and T genes. The sequence analysis indicates that the K and the two T genes are extremely homologous at the 5' portions as well as the 5'-flanking regions of at least 1.0 kilobase pairs. The S1 nuclease and primer extension analyses show that both T and K mRNAs start with three sets of the homologous initiation sites, and the utilization of the two 3'-side initiation sites of the T genes markedly increases under inflammation conditions. In contrast, no such elevation is observed for the three initiation sites of the K gene. Thus, although the sequences involved as transcription initiation sites are extremely homologous, their utilizations in the expressions of the K and T genes are regulated differently under inflammation conditions.

Tissue levels of atrial natriuretic polypeptide (ANP) messenger RNA (ANPmRNA) and ANP in the rat heart were measured simultaneously. In Wistar rats, ANPmRNA of the same size (approximately 0.95 kbp) was detected in all four chambers of the rat heart. The ANPmRNA level was the highest in the right atrium, and the left atrial level was slightly lower than the right atrial level. Ventricular levels were more than two orders of magnitude lower than atrial levels. Tissue ANP concentrations of four chambers were roughly parallel to ANPmRNA levels. In spontaneously hypertensive rats (SHR) with the elevated plasma ANP level, the ANPmRNA level in the left atrium was substantially increased. The left/right ratio of atrial ANPmRNA level in SHR (150%) was higher than that in control Wistar Kyoto rats (WKY) (90%). In contrast, the left/right ratio of atrial ANP concentration was decreased in SHR (44%) compared with that in WKY (84%). The ratio of ANP to ANPmRNA levels in the left atrium of SHR was about three times smaller than that in the right atrium of SHR, and those in bilateral atria of WKY. These results indicate that the biosynthesis and secretion of ANP from the left atrium is preferentially increased in SHR. Thus, simultaneous determination of ANPmRNA and ANP levels is a refined strategy of investigation for the biosynthesis, storage and secretion of ANP.

Tissue levels of atrial natriuretic polypeptide (ANP) and ANP messenger RNA (mRNA) in the atrium and ventricle were measured simultaneously in spontaneously hypertensive rats (SHR) and its substrain, SHR-stroke prone (SHRSP), and these levels were compared with those in control Wistar-Kyoto rats (WKY). At 27 weeks of age with established hypertension and ventricular hypertrophy, ANPmRNA levels in ventricles from SHR and SHRSP were markedly increased, and total contents of ventricular ANPmRNA in SHR and SHRSP were approximately 50% and 250%, respectively, of the corresponding atrial contents. However, levels and total contents of atrial ANPmRNA in SHR and SHRSP were similar to those of WKY, and the total content of ventricular ANPmRNA in WKY was only 6% of the content of atrial ANPmRNA. ANP concentrations in ventricles of SHR and SHRSP were increased in association with the augmentation of ANPmRNA levels. During the prehypertensive stage at 6 weeks of age, slight increases in levels and total contents of ANPmRNA and ANP in the ventricle were observed only in SHRSP. These results demonstrate that the expression of the ANP gene is markedly augmented in ventricles of SHR and SHRSP, especially of SHRSP, at the stage of established hypertension and ventricular hypertrophy, and they also suggest that these genetically hypertensive rats are one of the best animal models to investigate the biosynthesis, storage, and secretion of ventricular ANP.

We have isolated the renin gene from a Sprague-Dawley rat genomic library and determined its complete nucleotide sequence. The single rat renin gene is approximately 11,000 bases in length and consists of nine exons and eight introns. The amino acid sequence predicted from the genomic sequence indicates that the rat renin precursor consists of 402 amino acid residues, and shows 85%, 82% and 68% homology to the mouse Ren-1 and Ren-2 , and human renins, respectively. The canonical promoter “TATA” boxes, TATAAA and TAATAA, are found 27 and 57 base-pairs upstream from the putative cap site, respectively. Several attractive regulatory sequences analogous to glucocorticoid, estrogen receptor-binding sites, cAMP-responsive element and SV40 enhancer core sequences were noted in the 5′-flanking region of the gene. In the first intron, segments with an average size of 38 base-pairs containing a NcoI site are present at 46 tandem repeats within 1710 base-pairs. A “CA” element consisting of (CA) 27 was identified in intron 3. Furthermore, intron 8 contains a sequence that shows about 93% homology to that of the neuronal identifier sequence.

In isolated rat hepatocytes, exposed to angiotensin II and glucocorticoids, angiotensinogen mRNA increased within 30-60 min, and angiotensinogen secretion with a time lag of about 2 hours. After 4 hours, angiotensinogen mRNA, estimated by liquid hybridization with radiolabeled cRNA, was 5.9 +/- 0.4 in control, and 10.8 +/- 0.8, 11.7 +/- 0.4, 16.1 +/- 0.8 and 21.7 +/- 0.2 pg/micrograms of total RNA in cells exposed to angiotensin II (9 nM and 90 nM), hydrocortisone (100 microM) and dexamethasone (10 microM) respectively. The corresponding secretion rates of angiotensinogen were 72 +/- 7, 124 +/- 4, 132 +/- 12, 220 +/- 19 and 217 +/- 18 fmol angiotensinogen/mg wet weight/hour. Thus, angiotensin II stimulates angiotensinogen synthesis and secretion by acting at a pretranslational site.

The primary structure of human renin precursor has been deduced from its cDNA sequence. A library of cDNA clones was constructed from human kidney poly(A)+ RNA by applying the vector/primer method of Okayama and Berg. The library was screened for human renin sequences by hybridization with the previously cloned mouse renin cDNA. Of the 240,000 colonies screened, 35 colonies that were positive for hybridization were isolated. Two recombinant plasmids containing long inserts of about 1,300 and 1,600 base pairs were selected for sequence analysis. The amino acid sequence predicted from the cDNA sequence shows that the human renin precursor consists of 406 amino acids with a pre and a pro segment carrying 20 and 46 amino acids, respectively. A high degree of sequence homology was found upon comparison of the mouse and human renins. Close similarities were also observed in the primary structures of renin and aspartyl proteinases that have known three-dimensional structures, suggesting a similar tertiary structure for renin.

The amino acid sequence for a novel human membrane protein that induces selective potassium permeation by membrane depolarization was deduced by molecular cloning and sequence analysis of its genomic DNA. This protein consists of 129 amino acid residues and shares several structural characteristics with the rat counterpart. These include a single putative transmembrane domain surrounded by many charged amino acid residues, two potential N-glycosylation sites at the amino-terminal portion and a single cysteine residue at the carboxyl-terminal portion. The transmembrane domain and its flanking carboxyl-terminal sequence are highly conserved between the human and rat sequences. Because the slowly activating potassium current elicited by the human protein on its expression in Xenopus oocytes is indistinguishable from that induced by the rat protein, the sequence conserved at the transmembrane domain and its following sequence should play an essential role in the induction of selective K+ permeation.

The transmitter receptor matches and mismatches in the angiotensin (ANG) immunoreactive (IR) neuronal systems of the rat CNS have been characterized in various regions by means of ANG II immunocytochemistry and 125I-angiotensin II receptor autoradiography. By means of in situ hybridization the distribution of angiotensinogen mRNA has been mapped out and related to the distribution of ANG IR. In some areas, high densities of ANG IR nerve terminals and ANG II receptors (e.g. paraventricular hypothalamic nucleus, locus coeruleus and nucleus tractus solitarius) or high densities of ANG II receptors alone (e.g. medial geniculate body, subthalamic nucleus and superficial layer of the superior collicle) were often associated with high levels of angiotensinogen mRNA, suggesting the existence of an extracellular formation of ANG II, mediating biological responses. These results underline a role of ANG peptides in volume transmission in addition to transmitter function. Other areas, such as nuc. n. hypoglossi, practically lacking ANG IR terminals, pericarya and receptors, also contained high levels of angiotensinogen mRNA, suggesting a different role of angiotensinogen in these areas. Evidence for presynaptic (turnover changes) and post-synaptic (receptor-receptor crosstalk) interactions with CA neuronal systems has been obtained especially in cardiovascular centers. Thus, ANG II reduces in a concentration related way the affinity of 3H-paraminoclonidine binding sites in the dorsomedial medulla without influencing the Bmax value. These results indicate the existence of intramembrane interactions between ANG II and alpha 2 adrenergic receptors. Finally paraventricular but not perifornical ANG immunoreactive nerve cells costore nuclear glucocorticoid receptor IR, suggesting that some ANG neurons may be directly regulated by glucocorticoids.

The expressions of the renin, angiotensinogen, and atrial natriuretic polypeptide (ANP) genes were studied in rats with varying sodium intakes. mRNAs for angiotensinogen and renin were measured by a sensitive radiodensitometric hybridization assay. ANP mRNA was determined by a dot-blot hybridization assay. In the high-sodium state, plasma renin concentration (PRC), renal renin concentration (RRC), and renal renin mRNA decreased by 88, 90, and 75%, respectively. In the low-sodium state, PRC, RRC, and renin mRNA increased 17-fold, 2.5-fold, and 4.5-fold, respectively. With captopril treatment during sodium depletion, PRC and renin mRNA increased 144-fold and 17.1-fold, respectively, and RRC increased 4.2-fold. The angiotensinogen mRNA level in the liver and kidney decreased in the high-sodium and increased in the low-sodium state to similar extents. Captopril treatment decreased angiotensinogen mRNA in both organs by about half. Plasma ANP and ANP mRNA decreased in the low- compared with the high-sodium state, whereas atrial ANP content was unaltered in both low- and high-sodium states. These results demonstrate that sodium intake affects the expression of the renin and angiotensinogen genes and slightly alters the expression of ANP gene.