Atrial mRNA Expression Research Articles

Protein kinase C-dependent regulation of the human AT1 promoter in vascular smooth muscle cells.

The expression level of angiotensin II (ANG II) type 1 receptors (AT1) determines the magnitude of ANG II signaling in vascular smooth muscle cells (VSMC). AT1 mRNA expression in cultured bovine VSMC increased twofold after 8 h of protein kinase C (PKC) activation with phorbol 12-myristate 13-acetate (PMA), whereas stimulation with forskolin did not alter the AT1 mRNA level. The expression of AT1 promoter/exon 1 [-513/+92 base pairs (bp)] luciferase constructs transfected into VSMC increased 2.4-fold with PMA stimulation. In-gel kinase assays demonstrated rapid phosphorylation of mitogen-activating protein kinases (MAPK) ERK1 and ERK2 by PMA. Electrophoretic gel mobility shift assays showed sequence-specific binding of nuclear proteins from PMA-activated VSMC, identified as activator protein 1 (AP-1) complex in competition assays, to a radiolabeled AT1-promoter fragment (-368/-399 bp). Recombinant AP-1 binds in a sequence-specific manner to the -386/-399-bp region. Site-specific mutagenesis destroying the AP-1 site, the adjacent polyoma enhancer activator 3 element, or both sites simultaneously indicated that both sites together are necessary and sufficient to control basal and PMA-induced activation of the human AT1 promoter in transfected VSMC. The capability of the phorbol ester PMA to activate the human AT1 promoter in VSMC via an AP-1 element suggests a prominent role for PKC/MAPK and Ets proteins in AT1 regulation.

Distinct mechanisms of modulation of angiotensin II type I receptor gene expression in heart and aorta.

The purpose of the present study was to test the hypothesis that hypertension induced by reduced renal mass (RRM) upregulates gene expression of the type 1 angiotensin II (Ang II) receptor (AT1) in the thoracic aorta and heart through an Ang II-dependent mechanism. Three groups of rats were given 1% NaCl water and subjected to RRM, RRM plus captopril (RRM+Cap, 30 mg/kg per day), or sham surgery. Tail-cuff systolic blood pressure was significantly elevated in RRM and RRM+Cap rats compared with sham-operated rats. The ratios of the medial wall area of the thoracic aorta and heart weight to body weight were significantly elevated in RRM and RRM+Cap rats compared with sham-operated rats. Northern blot analysis indicated that the ratio of AT1 to GAPDH mRNA in the aorta was significantly higher in RRM (1.85 +/- 0.52) compared with sham-operated (0.21 +/- 0.04) and RRM+Cap (0.55 +/- 0.20) rats. In contrast, the ratio of AT1 to GAPDH mRNA in the heart was significantly increased in both RRM (1.09 +/- 0.23) and RRM+Cap (1.00 +/- 0.09) compared with sham-operated (0.34 +/- 0.06) rats. Thus, RRM hypertension upregulates AT1 mRNA expression in both the hypertrophied aorta and heart. Captopril treatment without altering blood pressure in RRM rats prevents the increase in AT1 mRNA in the aorta but not the heart. These results suggest that different tissue-specific mechanisms of AT1 gene regulation exist; ie, in aorta, an Ang II-or kinin-dependent mechanism is operant, whereas in heart, RRM-induced upregulation of AT1 mRNA may be pressure dependent.

Influence of AT1 Receptor Activity on AT1 and AT2 Receptor Expression During Fetal Life. † 284

The present study was designed to test the hypothesis that an increase in AT1 receptor activity participates in the decline in AT2 mRNA expression associated with fetal maturation (Pediatr Res 38:896904, 1995). To test this hypothesis, we studied eleven pairs of chronically instrumented twin fetal sheep at 95d gestation and seven pairs at 110d gestation (term is 145d). One twin received intravenous infusion of losartan (L) (10mg/kg bolus followed by 20 μg/kg/min for 48 hr) while the other served as a salinetreated control (C). In the 95d animals, no change in heart rate, mean arterial blood pressure (MABP), plasma renin activity (PRA), or aldosterone levels were observed during L infusion. Furthermore, L did not alter AT1 and AT2 mRNA expression in 95d fetuses; AT1 and AT2 mRNA levels were consistent in C and L animals kidney cortex, kidney medulla, liver, and adrenal. In contrast, in 110d fetuses, L decreased MABP from 40.1±1mmHg to 33±1mmHg(p<0.001) while PRA increased from 4.8±1.5 to 9.8±3 ngAI/ml/hr (p<0.05). In addition, L decreased 110d kidney medulla AT1 by 32±12% (p<0.05) and reduced kidney medulla AT2 by 32±14%(p<0.05) mRNA expression. Likewise, adrenal AT2 fell by 14±11%. To determine if these changes in mRNA expression during L were secondary to changes in MABP, three additional sets of 110d twin fetuses were studied; one twin received L while the other twin received L plus phenylephrine to keep MABP from falling. When compared to L fetuses, keeping MABP normal prevented changes in AT1 and AT2 mRNA levels.

Expression and localization of angiotensin II type 1 receptor mRNA in rat ocular tissues.

Angiotensin II type 1 receptor (AT1) is expressed in a variety of tissues. However, the localization of the specific AT1 mRNA in several ocular tissues has not been fully elucidated. In the present study, we investigated the expression and distribution of AT1 mRNA in the several ocular tissues of Wistar-Kyoto rats (WKYs) using the technique of reverse transcription with polymerase chain reaction. The 5 cornea, sclera, iris, ciliary body, and choroid samples tested were negative. When we analyzed 5 retina samples, 4 were positive. AT1 mRNA was more significantly expressed in the retina of WKYs than in the other ocular tissues of WKYs (p = 0.0238 < 0.05). Our study provides the first direct evidence that the AT1 is abundantly present in the WKY retina portions. This is a strategic location for an ocular renin-angiotensin system to influence retinal cicrulation.

TISSUE-SPECIFIC AND DIFFERENTIAL REGULATION OF AT1 AND AT2 GENE EXPRESSION FOLLOWING ADMINISTRATION OF AN AT1 ANTAGONIST DURING FETAL LIFE. † 2186

Fetal kidney AT2 mRNA levels are elevated early in gestation and decrease with fetal maturation, whereas AT1 mRNA expression increases rapidly before birth. The present study was designed to determine if the developmental rise in AT1 expression contributes to the decrease in AT2 expression with advancing gestational age. To test this hypothesis, eight pairs of chronically-instrumented twin fetal sheep (111±2 d gestation, term 145 d) were studied; one of the fetuses received an intravenous infusion of the AT1 antagonist losartan (L) (20μg/kg/min for 48 h) while the other served as a saline-treated control (C)(0.9% NaCl at 0.1 ml/h). Mean arterial blood pressure did not change in C, but decreased from 40±1 to 33±2 mmHg in L-treated fetuses(p<0.01). Heart rate did not change in C or L fetuses. Plasma renin activity increased significantly (p<0.01) in L-treated fetuses from 4.3±1.3 to 10.7±3.8 ng AI/ml/h. L produced significant(p<0.05) decreases in kidney medulla AT1 (-32.4±12.6%) and AT2 mRNA levels (-7.4±2.9%) but did not affect AT1 or AT2 expression in the kidney cortex. In the adrenal, L did not alter AT1 but increased AT2 expression by 246±130% (p<0.05). L did not alter AT1 or AT2 expression in fetal liver. The present results demonstrate that inhibition of AT1 receptor activity produces tissue-specific and differential regulation of both AT1 and AT2 genes during fetal life, and that the decrease in kidney AT2 expression during fetal development is not dependent on a rise in AT1 receptor activity.

Renin and Angiotensin II Receptor Gene Expression in Kidneys of Renal Hypertensive Rats

The aim of this investigation was to examine the interrelation between renal mRNA levels of renin and angiotensin II receptor type 1 (AT1) in a renin-dependent form of experimental hypertension. Rats were studied 4 weeks after unilateral renal artery clipping. Mean blood pressure and plasma renin activity were significantly higher in the hypertensive rats (n = 10 206 +/- mm Hg and 72.4 +/- 20.9 ng/mL-1/h-1, respectively) than in sham-operated controls (n = 10, 136 +/- 3 mm Hg and 3.3 +/- 0.5 ng/mL-1/h, respectively). Northern blot analysis of polyA+ RNA obtained from the kidneys of renal hypertensive rats showed increased levels of renin mRNA in the clipped kidney, whereas a decrease was observed in the unclipped kidney. Plasma renin activity was directly correlated with renin mRNA expression of the poststenotic kidney (r = .94, P < .01). AT1 mRNA expression was lower in both kidneys of the hypertensive rats. This downregulation was specific for the AT1A subtype since the renal expression of the AT1B subtype remained normal in hypertensive rats. The downregulation of the renal AT1A receptor may be due to high circulating angiotensin II levels. This is supported by the significant inverse correlation (r = .71, P < .01) between plasma renin activity and AT1A mRNA expression measured in the clipped kidney of the hypertensive rats.

Ontogenic Changes and Regulation of Renal Angiotensin II Type 1 Receptor Gene Expression during Fetal and Newborn Life

Factors regulating the expression of the angiotensin II subtype 1 (AT1) receptor during fetal life have not been investigated previously. The present study was designed 1) to characterize the ontogeny of AT1 receptor gene expression in the kidney of fetal and newborn sheep and 2) to determine the influence of both glucocorticoids and renal nerves in modulating AT1 gene expression during fetal life and during the transition from fetal to newborn life. We first isolated and cloned a PCR product that has 98 and 94% homology with the cDNA encoding the bovine and pig AT1 receptors, respectively, and 99 and 98% homology with the corresponding deduced protein sequences. Probing with this cDNA, we demonstrated that renal AT1 mRNA expression did not change significantly during the last trimester of gestation in fetal sheep or immediately after birth but decreased significantly 10 d after birth. We also demonstrated that renal denervation in the fetus had no effect on renal AT1 gene expression in 24-h-old newborn lambs. On the other hand, we observed in 130-d twin fetuses that a continuous intraperitoneal infusion (1 mL/h) of cortisol (3 mg/h or 6.2 mumol/h) for 48 h in one of the twins increased the fetal plasma cortisol concentration from 32.0 +/- 7.1 to 1126 +/- 231 nmol/L and produced a significant decrease (p < 0.005) in renal AT1 gene expression compared with the control twin receiving an intraperitoneal infusion of 0.9% NaCl. In summary, this study demonstrates that renal AT1 gene expression is elevated during fetal life and decreases after birth. It is also shown that glucocorticoids, but not renal nerves, contribute to the regulation of renal AT1 gene expression during development.

Cardiac and plasma atrial natriuretic peptide after 9-day hindlimb suspension in rats.

To determine atrial natriuretic peptide (ANP) adaptation to simulated weightlessness, immunoreactive plasma (ir-NH2- and ir-COOH-terminals) and atrial (ir-COOH-terminal) ANP levels, atrial mRNA expression, immunoreactive cardiocyte ANP levels (ir-NH2- and ir-COOH-terminals), and ultrastructural observations of granules in atrial cardiocytes were assessed in male Wistar rats after a 9-day hindlimb suspension. Plasma ir-NH2- and ir-COOH-terminal ANP concentrations decreased by 17 (P < 0.05) and 37% (P < 0.05), respectively, in suspended rats. A concomitant ir-COOH-terminal ANP content reduction was also observed in left (31%; P < 0.01) and right atria (25%; P < 0.05). Atrial ANP mRNA expression was severely depleted in the right atrium and less so in the left atrium after 9 days of hindlimb suspension. Immunocytochemistry observations demonstrated lowered NH2- and COOH-terminal ANP immunoreactivities in left and right atria from suspended rats. A reduced number of storage granules (dense granules) in both atria was also noted on ultrastructural analysis. It was concluded that ANP biosynthesis, storage, and release were decreased after a 9-day hindlimb suspension.