Adenosine A1 receptor mRNA in microdissected rat nephron segments.

Adenosine plays several roles in the kidney mediated by the specific receptors A1, A2, and possibly A3. We studied the localization of adenosine A1 receptor mRNA in rat nephron segments using reverse transcription and polymerase chain reaction (RT-PCR). The nephron segments of male Sprague-Dawley rats (6 to 8 weeks old) were microdissected. Total RNA was prepared by the acid-guanidinium-phenol-chloroform method and used in the following RT-PCR assay. Because the PCR primers spanned no intron, samples reacted in the absence of RT were used as controls for amplification of genomic DNA. The PCR products were size-fractionated by electrophoresis, visualized with ethidium bromide staining, and confirmed by Southern blot analysis. PCR products were detected in all of the nephron segments examined. No signals were detected in samples reacted in the absence of RT. Strong signals were detected in glomeruli, medullary collecting duct, cortical thick ascending limb, and medullary thick ascending limb, while weak signals were found in proximal convoluted and straight tubules. Previously, the presence of A1 receptors has been demonstrated in glomeruli, collecting duct, and thick ascending limb in the rat kidney by autoradiography and binding studies. In addition to these segments, we further detected A1 receptor mRNA in proximal convoluted and straight tubules. Thus, A1 receptor mRNA seems to be broadly expressed along the nephron.

Essential hypertension and 5' upstream core promoter region of human angiotensinogen gene.

The angiotensinogen (AGT) gene M235T variant is associated with essential hypertension and elevated plasma AGT concentrations, although the underlying mechanisms are unknown. Recent studies have suggested that AGCE 1 (human AGT gene core promoter element 1) located in the 5' upstream core promoter region (position -25 to -1) of the human AGT gene has an important part in the expression of AGT mRNA by binding with transcription factor AGCF 1 (human AGT gene core promoter element binding factor 1), and a mutation at -20 from adenine to cytosine (A-20C) increases the level of expression of this transcript. We therefore examined subjects with this mutation to study the association with increased plasma AGT concentrations and with essential hypertension. One hundred eighty-eight subjects receiving no antihypertensive medication were examined with regard to the correlation between A-20C and plasma AGT concentrations, and 234 subjects were studied with respect to the association between A-20C and essential hypertension. A-20C was determined by polymerase chain reaction-restriction fragment length polymorphism analysis with EcoOR 109I. Multiple regression analysis showed a weak but significant correlation between A-20C and plasma AGT concentrations (P=.047) and essential hypertension (P=.049). The results suggest that A-20C may underlie the increase in plasma AGT concentrations and be involved in the development of essential hypertension.

Tissue angiotensinogen gene expression induced by lipopolysaccharide in hypertensive rats.

There is now convincing evidence that various tissues express their own tissue renin-angiotensin system, which may be regulated independently of the systemic renin-angiotensin system. However, little information is available on the regulation of the tissue renin-angiotensin system. We investigated the regulation of tissue angiotensinogen gene expression with respect to the development of hypertension. We measured basal and lipopolysaccharide-stimulated plasma angiotensinogen concentrations by radioimmunoassay and examined the expression of tissue angiotensinogen by Northern blot analysis in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) at 4 and 13 weeks of age. Basal plasma angiotensinogen concentration in SHR was comparable to that in WKY at 4 weeks of age and was significantly higher than that in WKY at 13 weeks of age. Lipopolysaccharide induced a significant increase in plasma angiotensinogen concentration in both WKY and SHR at 4 and 13 weeks of age. At 4 weeks of age, the basal levels of angiotensinogen mRNA in the liver, fat, adrenal, and aorta were higher in WKY than in SHR. At 13 weeks of age, the basal levels of angiotensinogen mRNA in the fat, adrenal, aorta, spleen, and kidney were higher in WKY than in SHR, while that in the liver did not differ significantly between the two strains. At 4 weeks of age, pretreatment with lipopolysaccharide increased the angiotensinogen mRNA levels in the liver, fat, adrenal, and aorta in both WKY and SHR. At 13 weeks of age, pretreatment with lipopolysaccharide increased the angiotensinogen mRNA levels in the liver, aorta, and adrenal; decreased those in the spleen; and had no effect in the kidney in both WKY and SHR. Interestingly, lipopolysaccharide increased the angiotensinogen mRNA level in fat only in SHR, with no effect in WKY, at 13 weeks of age. Lipopolysaccharide stimulated tumor necrosis factor-a mRNA expression in fat of WKY and SHR, and the increase in tumor necrosis factor-alpha mRNA level in SHR was significantly greater than that in WKY. Therefore, the increased tumor necrosis factor-alpha mRNA expression may be involved in the increased lipopolysaccharide-induced expression of angiotensinogen gene in fat of SHR at 13 weeks of age. These data suggest that the transcriptional and probably posttranscriptional regulation of angiotensinogen mRNA differs between SHR and WKY, that the regulation of angiotensinogen gene expression is tissue-specific, and that the altered expression of the angiotensinogen gene may be involved in the development of hypertension.

Effect of genetic deficiency of angiotensinogen on the renin-angiotensin system.

This study examined expression of renin-angiotensin system (RAS) component mRNAs in angiotensinogen gene knockout (Atg-/-) mice. Wild-type (Atg+/+) and Atg-/- mice were fed a normal-salt (0.3% NaCl) or high-salt (4% NaCl) diet for 2 weeks. Angiotensinogen, renin, angiotensin-converting enzyme (ACE), angiotensin II type la receptor (AT1A), and angiotensin II type 2 receptor (AT2) mRNA levels were measured by Northern blot analysis. In Atg+/+ mice, activities of circulating RAS and renal angiotensinogen mRNA level were decreased by salt loading, whereas levels of renal and cardiac ACE; renal, brain, and cardiac AT1A; and brain and cardiac AT2 mRNA were increased by salt loading. Although activities of circulating RAS were not detected in Atg-/- mice, salt loading increased blood pressure in Atg-/- mice. In Atg-/- mice, renal renin mRNA level was decreased by salt loading; in contrast, salt loading increased renal AT1A and cardiac AT2 mRNA levels in Atg-/- mice, and these activated levels in Atg-/- mice were higher than those in Atg+/+ mice fed the high-salt diet. Thus, expression of each component of the RAS is regulated in a tissue-specific manner that is distinct from other components of systemic and local RAS and that appears to be mediated by a mechanism other than changes in the circulating or tissue levels of angiotensin peptides.

Increased cardiac angiotensin II receptors in angiotensinogen-deficient mice.

Two subtypes of angiotensin II (Ang II) receptors, type 1 (AT1-R) and type 2 (AT2-R), have been identified in the heart. However, little is known about the regulation of cardiac AT1-R and AT2-R by Ang II in vivo. Thus, we examined cardiac AT1-R and AT2-R in angiotensinogen-deficient (Atg-/-) mice that are hypotensive and lack circulating Ang II. Cardiac Ang II receptors (Ang II-R) were assessed by radioligand binding with 125I-[Sar1,Ile8]-Ang II in plasma membrane fractions. AT1-R and AT2-R were distinguished using their specific antagonists CV-11974 and PD123319, respectively. Total densities of Ang II-R and AT1-R density were significantly greater in the Atg-/- mice than Atg+/+ mice (31.1+/-2.8 versus 18.8+/-2.1, 28.7+/-3.0 versus 16.9+/-2.3 fmol/mg protein, P<.01, respectively), and AT2-R showed a slight but not significant increase in Atg-/- mice relative to Atg+/+ control animals. Kd values were not different between the two groups. In contrast to binding experiments, levels of Ang II type 1a receptor (AT1a-R) and AT2-R mRNA did not differ between Atg-/- and Atg+/+ mice. These results suggest that lack of Ang II may upregulate AT1-R through translational and/or posttranslational mechanisms in Atg-/- mice.

Tissue-specific regulation of angiotensinogen gene expression in spontaneously hypertensive rats.

Angiotensinogen is expressed in many tissues besides the liver. Recent studies have suggested that abnormalities in the regulation of angiotensinogen gene expression may be involved in the development of hypertension. However, little information is available concerning the functional significance of tissue angiotensinogen. In this study, we measured plasma angiotensinogen concentration by radioimmunoassay and examined the expression of tissue angiotensinogen by Northern blot analysis in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Although plasma angiotensinogen concentration in SHR was comparable to that in WKY at 6 weeks of age, it was increased significantly at 14 weeks of age in SHR and became higher than that in WKY. The levels of hepatic angiotensinogen mRNA were similar in SHR and WKY, and the levels of aortic, adrenal, and renal angiotensinogen mRNAs were lower in SHR than in WKY at both 6 and 14 weeks of age. Brain angiotensinogen expression in SHR was higher than in WKY at 6 weeks of age and was comparable to that in WKY at 14 weeks of age. On the other hand, cardiac and fat angiotensinogen mRNA levels were significantly increased at 14 weeks of age in SHR. These results demonstrate that the expression of tissue angiotensinogen is regulated differently in SHR and WKY and indicate that the development of hypertension is accompanied at least temporally with increases in plasma angiotensinogen concentration as well as cardiac and adipogenic angiotensinogen mRNA in SHR.

Endothelial nitric oxide synthase gene polymorphism and acute myocardial infarction.

Recently a point mutation of guanine to thymine at nucleotide position 1917 in the endothelial nitric oxide synthase (eNOS) gene has been reported to be associated with coronary artery spasm. In addition, a significant association of the 4a/b polymorphism in intron 4 of the eNOS gene with coronary artery disease has been reported. However, the implications of these polymorphisms with respect to acute myocardial infarction (AMI) remain to be established. We conducted a case-control study of 226 patients with AMI and 357 healthy gender- and age-matched control subjects. In the former group, coronary angiograms were evaluated according to angiographic criteria based on the number of diseased vessels (>/=75%) and the number of stenotic lesions (>/=50%). Homozygosity for the Glu-Asp298 polymorphism existed in 5 of 226 patients with AMI (2.2%) but not in any of the 357 control subjects (P=.0085). However, when we evaluated the coronary angiograms of 226 case patients, there was no difference in the number of diseased vessels or the number of stenotic lesions between the patients with this homozygote and those without it. By contrast, there was no evidence of a significant increase in the risk of AMI or the severity of coronary atherosclerosis among individuals with the a/a genotype of the eNOS4a/b polymorphism. Our results imply that patients who are homozygous for the Glu-Asp298 polymorphism may be genetically predisposed to AMI; however, this mutation apparently is not related to the severity of coronary atherosclerosis. Further studies are needed to confirm our results and characterize the molecular mechanisms by which eNOS is involved in susceptibility to AMI.

Endocrinological abnormalities in angiotensinogen-gene knockout mice: studies of hormonal responses to dietary salt loading.

OBJECTIVE: Physiological roles of the renin-angiotensin system in maintaining blood pressure and sodium-water balance in angiotensinogen gene-knockout mice were evaluated with special reference to endogenous pressor substances. METHODS: Angiotensinogen-gene knockout mice and control mice were fed a 0.3 or 4% NaCl diet for 2 weeks. Systolic blood pressure and urinary excretions of electrolytes, creatinine, aldosterone, adrenaline, noradrenaline, dopamine and vasopressin were measured. RESULTS: About 60% of our angiotensinogen-gene knockout mice did not survive until weaning. These mice presented with hypotension and polyuria. Urinary excretion of aldosterone from such mice was significantly lower (not detected) than that from control mice (2.0+/-0.3 pg/mg creatinine). In contrast, urinary excretion of vasopressin from angiotensinogen-gene knockout mice (0.7+/-0.1 ng/mg creatinine) was greater than that from control mice (0.3+/-0.1 ng/mg creatinine), and those of adrenaline and of noradrenaline were similar for knockout and control mice. After salt loading (a 4% NaCl diet), angiotensinogen-gene knockout mice exhibited a significant increase in systolic blood pressure (from 68.3+/-2.9 to 95.9+/-5.9 mmHg), significant decreases in urinary excretions of adrenaline (from 65+/-8 to 40+/-7 pg/mg creatinine) and noradrenaline (from 467+/-48 to 281+/-41 pg/mg creatinine) and no change in excretion of vasopressin compared with such mice fed a 0.3% NaCl diet CONCLUSION: The present results with angiotensinogen-gene knockout mice confirm that the renin-angiotensin system plays fundamental roles in maintaining the blood pressure and sodium-water balance. Because the vasopressin and catecholaminergic systems may be altered by lack of angiotensin in angiotensinogen-gene knockout mice, these systems perhaps are not able to restore blood pressure and sodium-water depletion to normal levels in these mice.

Analysis of molecular heterogeneity of Dahl/Iwai salt-sensitive rats and salt-resistant rats.

Molecular evidence, using DNA fingerprint analyses, of extensive genetic heterogeneity between spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and even within some of the WKY colonies has been reported. Thus we investigated the genetic relations between Dahl S and R rats newly inbred by Dr. Iwai. Genomic DNA was isolated from the liver of four Dahl S and four Dahl R rats, digested with the restriction enzyme HinfI or AluI, and separated in 1.2% agarose gel by electrophoresis. Then, DNA fingerprinting was performed by Southern blot analysis using the human myoglobin 33.6 minisatellite probe. Bands were detected in an alkaline phosphatase reaction system. Within the same strains, there was no heterogeneity of these fingerprinting patterns. The S and R rats shared 82% of the bands in the HinfI-digested DNA and 93% of those in the AluI-digested DNA. These shared values were much greater than the reported value (54%) between SHR and WKY from Charles River Laboratories. These newly inbred Dahl S and R rats may be appropriate, although still limited, experimental animals for investigating the pathophysiology of salt-sensitive hypertension.

Plasma angiotensinogen concentrations in obese patients.

A close relationship between obesity and hypertension has been recognized, and plasma angiotensinogen concentrations (p-AGT) have been reported to correlate with blood pressure (BP). However, little is known about AGT in obese patients with hypertension. To define the role of AGT in obese hypertension, we measured p-AGT in obese patients. The subjects were 42 obese patients diagnosed on the basis of a body mass index (BMI) of more than 25 kg/m2, and 21 sex- and age-matched nonobese patients, whose BMI was less than 25 kg/m2. The hypertensive patients had not previously received antihypertensive drugs. P-AGT (P < .05) and mean BP (P < .0001) was increased in the obese patients as compared with the nonobese patients. Positive correlations were observed between BMI and p-AGT, mean BP and p-AGT, and BMI and mean BP (all P < .05). However, after adjustment for blood pressure, p-AGT was not different between groups, and after adjustment a positive correlation remained only between BMI and mean BP. These results suggested the possible involvement of increased p-AGT in hypertension in obese patients, although this may be a secondary change to hypertension or obesity.

Developmental changes in expression of angiotensinogen mRNA in rat nephron segments.

We studied the localization of angiotensinogen mRNA in rat nephron segments and the differences in angiotensinogen mRNA levels between male Sprague-Dawley rats at 6 and 12 wk of age using reverse transcription and polymerase chain reaction (RT-PCR). Each nephron segment of the rat kidney was microdissected. Total RNA was prepared and used in the following RT-PCR assay. The PCR products were size-fractionated by agarose gel electrophoresis, visualized with ethidium bromide staining, and identified by Southern blot analysis. The relative amounts of products were determined by densitometry. Strong bands corresponding to angiotensinogen mRNA were detected from proximal convoluted and straight tubules, and weaker bands were found in glomeruli. The signals in all tissues in 12-wk-old rats were weaker than those in 6-wk-old rats. Since local angiotensinogen is the unique substrate of the tissue renin-angiotensin system and exerts an autocrine-paracrine influence on renal function, the changes in tubular angiotensinogen may be related to physiological and morphological changes in the rat kidney during development.

Relationship between hepatic angiotensinogen mRNA expression and plasma angiotensinogen in patients with chronic hepatitis.

Recent association and linkage studies suggested that angiotensinogen may play an important role in the pathogenasis of essential hypertension. However, there is little information in human concerning a relationship between plasma angiotensinogen levels and the angiotensinogen mRNA expression in the liver, which is the main production site of angiotensinogen. Therefore, the aim of this study was to examine whether hepatic angiotensinogen gene expression determines the level of circulating angiotensinogen and the activity of the renin-angiotensin system in humans. The subjects were 36 patients with chronic hepatitis. Blood was collected from each patients for estimation of plasma renin activity, plasma angiotensinogen and angiotensin II concentrations and several parameters of liver function. In addition, total RNA was isolated from liver biopsy specimens, which were then used to measure angiotensinogen mRNA with Northern blot analysis. Levels of angiotensinogen mRNA were detected easily in the liver biopsy specimens in all of the patients. Hepatic angiotensinogen mRNA levels were positively correlated with plasma angiotensinogen levels (r=0.41, P=0.013). In contrast, hepatic angiotensinogen mRNA levels did not show any significant relationship with plasma renin activity, plasma angiotensin II concentration, histological subgroup of hepatitis, histological activity index and parameters of liver function tests. The present study demonstrated, for the first time, that hepatic angiotensinogen mRNA levels correlated with plasma angiotensinogen concentration in humans.

'Tako-Tsubo' transient ventricular dysfunction: a case report.

During admission for investigation of dysphagia, an 82-year-old woman suddenly complained of dyspnea, which was followed by cardiogenic shock. Her symptoms, electrocardiogram, echocardiogram and laboratory data were compatible with an extensive acute anterior myocardial infarction. Emergency cardiac catheterization showed no atheromatous narrowing in any coronary artery. However, the contractions of the left and right ventricles were diffusely and severely impaired, except for some hyperkinesis of the basal area. The asynergy, as well as the abnormalities on the ECG, improved almost to normal by the 35th hospital day. An endomyocardial biopsy from the right ventricle during the acute phase showed atypical myocardial damage with proliferation of fine collagen fibers and small round-cell infiltration including polymorphologic leukocytes. This type of transient cardiac disorder has recently been described in Japan, and is called 'Tako-tsubo cardiomyopathy' because of the characteristic appearance of the left ventricular asynergy. In the present case, ventricular asynergy was not limited to the left ventricle, but was also present in the right ventricle.

Distribution of alpha 1B-adrenergic receptor mRNA expression along rat nephron segments.

Although several alpha-adrenergic receptor genes are expressed in the rat kidney, little information is available on their expression in the renal nephron segments. We investigated the distribution of alpha 1B-adrenergic receptor mRNA in rat nephron segments using reverse transcription and polymerase chain reaction (RT-PCR). The nephron segments of six- to eight-week-old male Sprague-Dawley rats were microdissected. Total RNA was prepared by the acid-guanidinium-phenol-chloroform method and used in the following RT-PCR assay. The PCR products were size-fractionated with electrophoresis, visualized with ethidium bromide staining and confirmed by Southern blot analysis. Because the PCR primers spanned an intron, the amplification product of the predicted size was considered to be from alpha 1B-adrenergic receptor cDNA and not from genomic DNA. The PCR products were detected in glomerulus (Glm), proximal convoluted and straight tubules (PCT, PST) and cortical and medullary thick ascending limbs of Henle (CTAL, MTAL). No signals were detected in cortical or medullary collecting ducts (CCD, MCD). Large signals were detected in the PCT, and PST, while small signals were found in the Glm, CTAL and MTAL. The alpha 1B-adrenergic receptor mRNA was detected for the first time in rat Glm, PCT, PST and TAL using RT-PCR. alpha 1BAR mRNA seems to be expressed in the specific sites along the nephron and may play significant roles in renal functions, although the specific physiological effects of the renal alpha 1B-adrenergic receptor are unknown.

Activation of angiotensinogen gene in cardiac myocytes by angiotensin II and mechanical stretch.

Circulating and cardiac renin-angiotensin systems (RAS) play important roles in the development of cardiac hypertrophy. Mechanical stretch of cardiac myocytes induces secretion of ANG II and evokes hypertrophic responses. Angiotensinogen is a unique substrate of the RAS. This study was performed to examine the regulation of the angiotensinogen gene in cardiac myocytes in response to ANG II and stretch. ANG II and stretch significantly increased the levels of angiotensinogen mRNA in cardiac myocytes. Actinomycin D completely inhibited ANG II- and stretch-mediated increases in angiotensinogen mRNA. Although CV-11974 abolished ANG II-mediated increases in mRNA level and promoter activity of the angiotensinogen gene, the inhibition of stretch-mediated activation by CV-11974 was significant but not complete. These results indicate that ANG II activates transcription of the angiotensinogen gene exclusively via ANG II type 1-receptor pathway and that stretch activates such transcription mainly via the same pathway in cardiac myocytes. Furthermore, factors other than ANG II may also be involved in stretch-mediated activation of the angiotensinogen gene in cardiac myocytes.

Possible role of c-Jun in transcription of the mouse renin gene.

BACKGROUND: Renin is a rate-limiting enzyme for activity of the circulating renin-angiotensin system (RAS) and expression of the renin gene is regulated by a variety of stimuli. In this study, we examined a possible role of c-Jun in the transcription of renin gene. METHODS: The renin promoter, chloramphenicol acetyltransferase (CAT), fusion genes with or without c-Jun expression vector (pSV-c-Jun) were transfected into human embryonic kidney (HEK) cells, and the effects of c-Jun were examined by deletion and mutation analyses of CAT assay and by in vitro transcription-primer extension assay. We also examined the effects of c-Jun on DNA-binding activity to the renin promoter by electrophoretic mobility shift assay (EMSA). Furthermore, we examined the effects of c-Jun on transcription of the renin gene in enriched juxtaglomerular (JG) cells by cotransfection with pSV-c-Jun and by treatment with antisense c-jun oligodeoxynucleotides. RESULTS: Promoter activity of the renin gene was increased by c-Jun overexpression in HEK cells, and the proximal promoter region from -47 to +16 was sufficient for transcriptional activation by c-Jun. Although mutation of activator protein-1 (AP-1) element-like sequences in the proximal promoter did not affect c-Jun-mediated stimulation, mutation of the core promoter including the TATA box inhibited c-Jun-mediated transcription. The results of EMSA showed that c-Jun overexpression produced a binding of nuclear factor, which was HEK cell-specific and distinct from TATA box-binding protein and AP-1 family transcription factor, to the renin core promoter region (RC element) from -36 to -20. The overexpression of c-Jun activated the renin promoter in renin-expressing JG cells, and antisense c-jun decreased the activity of renin promoter and expression of renin mRNA in JG cells. CONCLUSIONS: These results indicate that the RC element plays a role in c-Jun-mediated transcriptional regulation of the renin gene in HEK cells, and suggest that c-Jun participates in the regulation of renin gene expression in JG cells of the kidney.

Modulation of tissue angiotensinogen gene expression in genetically obese hypertensive rats.

Wistar fatty rats (WFR) show obesity and obesity-related features, including hypertension. In this study, we examined the expression of angiotensinogen mRNA in a variety of tissues at different times in WFR and control Wistar lean rats (WLR). WFR were obese and hypertensive at 16 and 24 wk. Plasma renin activity and plasma angiotensinogen concentration showed age-dependent increases in WFR but decreases in WLR. Northern blot analysis showed no significant differences in the levels of hepatic and renal angiotensinogen mRNA between WFR and WLR, and the levels of fat and adrenal angiotensinogen mRNA were lower in WFR than in WLR. On the other hand, the levels of cardiac angiotensinogen mRNA at 16 and 24 wk and those of aortic angiotensinogen mRNA at 16 wk were significantly higher in WFR than in WLR. These results show that the expression of tissue angiotensinogen mRNA is regulated differently in WFR and WLR and indicate that the development of hypertension in WFR is accompanied at least temporally with increases in plasma angiotensinogen concentration as well as in cardiac and aortic angiotensinogen mRNA. Moreover, these results suggest the existence of obesity hypertension-linked and tissue-specific regulation of angiotensinogen gene expression.

Regulation of cardiac angiotensinogen mRNA in vivo and in vitro.

In this study, to investigate the mechanism of hypertension-associated induction of cardiac angiotensinogen in vivo and in vitro, we studied the regulation of angiotensinogen mRNA in the hearts of genetically hypertensive rats and in the rat cardiomyocytes. Levels of cardiac angiotensinogen mRNA were significantly increased in the hypertensive rats. Steady state mRNA levels for angiotensinogen mRNA in cardiomyocytes were increased by angiotensin II and mechanical stretch. The addition of an angiotensin II type 1 receptor antagonist (CV11974) and a transcriptional inhibitor (actinomycin D) completely blocked the induction of angiotensinogen mRNA by angiotensin II in cardiomyocytes. The addition of CV11974 significantly, but not completely, inhibited the induction of angiotensinogen mRNA by mechanical stretch. Actinomycin D completely blocked the induction of angiotensinogen mRNA by stretch in cardiomyocytes. An angiotensin II type 2 receptor antagonist (PD123319) and a protein synthesis inhibitor (cycloheximide) did not affect the induction. These results indicate that the expression of cardiac angiotensinogen mRNA is activated by the development of hypertensive cardiac hypertrophy, and that angiotensin II and mechanical stretch activates the angiotensinogen gene via the angiotensin II type 1 receptor-pathway in cardiomyocytes.

A novel proximal element mediates the regulation of mouse Ren-1C promoter by retinoblastoma protein in cultured cells.

The protein product of the retinoblastoma susceptibility gene, RB, is a nuclear phosphoprotein that modulates transcription of genes involved in growth control via interactions with transcription factors. Renin is a rate-limiting enzyme of the renin-angiotensin system that regulates blood pressure and water-electrolyte balance. Renin gene expression is regulated in a tissue-specific and developmentally linked manner. Similarly, the expression of RB is controlled in a differentiation-linked manner. Thus, to investigate whether RB is involved in the regulation of renin gene expression, we examined the effects of RB on transcriptional activity of the mouse renin (Ren-1C) promoter. The Ren-1C promoter contains two transcriptionally important elements; the RU-1 (-224 to -138) and RP-2 (-75 to -47) elements. RB activated the Ren-1C promoter in human embryonic kidney cells. The promoter element responsible for RB-mediated transcriptional regulation was the RP-2 element. The results of DNA-protein binding experiments showed that RB increased nuclear binding activity to the RP-2 element, and site-directed mutation which disrupted binding of nuclear factors to the RP-2 element markedly reduced RB-mediated activation of Ren-1C promoter in human embryonic kidney cells. These results indicate that the RP-2 element plays an important role in RB-mediated transcriptional regulation of Ren-1C promoter activity in human embryonic kidney cells, thereby suggesting an interesting mechanism by which RB may modulate the renin-angiotensin system.