Angiotensin II maintains, but does not mediate, isoproterenol-induced cardiac hypertrophy in rats.

The role of angiotensin II (ANG II) in the development of isoproterenol (Iso)-induced cardiac hypertrophy was examined in rats. Iso increased cardiac mass, left ventricular RNA-to-DNA ratio, and the cardiac content of both myosin heavy chain and hydroxyproline in a dose-dependent manner, indicating that Iso-induced cardiac hypertrophy involves growth of both muscle and connective tissue. Cardiac hypertrophy reverted within 11-14 days after cessation of Iso. Propranolol prevented development of Iso-induced cardiac hypertrophy but did not affect the rate of its reversal. The ANG II receptor blocker losartan (Los) did not significantly decrease the hypertrophic response to Iso. Los injected after cessation of Iso dramatically enhanced the reversal of cardiac hypertrophy, even in rats that received Los with Iso during the induction of Iso-induced cardiac hypertrophy. ANG II, injected continuously at a subpressor dose that did not affect heart weight when given alone, inhibited reversal of cardiac hypertrophy when given after cessation of Iso. Los did not significantly affect the induction of the protooncogene c-fos by Iso. We conclude that endogenous ANG II has a major function in maintaining Iso-induced cardiac hypertrophy but does not mediate its induction. This suggests that different interactive stimuli may be required for development of cardiac hypertrophy, i.e., for initiation and for maintenance.

Rare renal transcription of the atrial natriuretic factor gene in rats. Demonstration by polymerase chain reaction.

Renal synthesis of a peptide homologous to atrial natriuretic factor (ANF) has been demonstrated. The aim of the present study was to determine if transcription of the ANF gene occurs in the kidney. Rat renal RNA was extracted from whole kidneys, and, separately, from the cortex and outer and inner medulla of rat kidneys. Probing with rat ANF-cDNA did not reveal a detectable message in Northern blot analysis, even when large quantities of RNA were used at low stringency hybridization conditions. Therefore, reverse transcription (RT) followed by 35 cycles of polymerase chain reaction (PCR) was used to search for the renal message for ANF. Two 21-mer primers encompassing the 450 base pairs (bp) of the coding region of the gene were used. Each cycle consisted of annealing at 56 degrees C, extension at 72 degrees C, and denaturation at 94 degrees C. The PCR product was proven to be identical to the ANF gene by high stringency hybridization, which revealed the expected 450-bp hybrid band. Furthermore, the sequence of this product was identical to that of the coding region of the ANF gene. We used an RNA-specific PCR to obtain this band as a single reaction product. We conclude that the transcript of the ANF gene exists in the kidney, at extremely low levels. The low abundance of the RNA message raises major concerns about its physiologic relevance. Direct evidence for the translation of this transcript, and its quantification and localization, is still required to determine its significance.

Pharmacokinetics of ANF and urodilatin during cANF receptor blockade and neutral endopeptidase inhibition.

Urodilatin is a new member of the family of natriuretic peptides. It is of renal origin. Previous reports indicate that urodilatin is natriuretic in lower doses than atrial natriuretic factor (ANF)-(99-126) and that it might be more effective than ANF in the treatment of cardiovascular edema. The present study was designed to compare the pharmacokinetics of the hydrolysis and clearance of 125I-labeled urodilatin and 125I-ANF. In control rats, the volume of distribution (Vss), metabolic clearance rate (MCR), and distribution half-life (distribution t1/2) of urodilatin in plasma were not significantly different from those of ANF. Infusion of clearance (c)ANF-(4-23), a specific ligand for receptors that clear ANF in excess amounts (i.e., a bolus injection of 100 micrograms/kg followed by a continuous infusion of 10 micrograms.kg-1 x min-1), increased the amount of intact peptide in the plasma to the same extent for both urodilatin and ANF. In addition, cANF-(4-23) decreased the Vss and the MCR and increased the distribution t1/2 of both peptides to about the same degree. Prior treatment of rats with SQ-28,603, a specific neutral endopeptidase (NEP; EN 3.4.24.11) inhibitor, was without significant effect on the metabolic clearance of urodilatin, whereas it decreased the clearance of ANF by 65%. Furthermore, an infusion of SQ-28,603 suppressed the appearance of the hydrolytic products of ANF in blood but not of urodilatin. Moreover, the inhibitor increased the total amount of ANF recovered in the kidneys to five times the control values, whereas it did not alter the renal uptake of urodilatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of riboflavin analogues and diuretics on the spontaneously hypertensive rat heart.

The chronic treatment of spontaneously hypertensive rats (SHR) with 7,8-dimethyl-10-(3-chlorobenzyl) isoalloxazine [CBI], 7,8-diethyl-10-aminol isoalloxazine [DEAI], enduron (methyclothiazide) and amiloride were studied for their effects on blood pressure and cardiac contractile protein ATPase activities. After 35 weeks of treatment all the above antihypertensive agents showed a decrease in blood pressure in the SHR (p less than 0.01). Chronic treatment with CBI, DEAI, enduron, and amiloride significantly improved the myofibrillar ATPase activity at all pCa2+ concentrations (p less than 0.01). Furthermore, CBI, DEAI, enduron, and amiloride drug treatments enhanced actin-activated myosin ATPase activity (p less than 0.01). The Ca2(+)-activated myosin ATPase activity was significantly elevated after treating with CBI and DEAI (p less than 0.01). These results suggest that the antihypertensive agents used in this study helped in reducing the blood pressure with a subsequent increase in myocardial contractile protein ATPase activity.

Atrial natriuretic peptide mRNA is regulated by glucocorticoids in vivo.

In these studies glucocorticoids were found to increase the plasma levels of atrial natriuretic peptide (ANP) as well as the expression of the ANP gene in the Sprague-Dawley rat. Plasma ANP rose two-fold after 48 hrs. of exposure to dexamethasone (1 mg/day) in both intact and adrenalectomized animals. This was accompanied by a 1.5-2.0 fold increase in the levels of atrial and ventricular ANP transcripts. Deoxycorticosterone acetate (5 mg/day), administered on the same schedule, failed to increase either plasma ANP levels or cardiac ANP mRNA accumulation. These effects suggest that ANP may have a potential role as a mediator of glucocorticoid activity in the cardiovascular system and support the hypothesis that ANP is a glucocorticoid-regulated gene.

Antihypertensive effect of riboflavin analogues in spontaneously hypertensive rats.

Our study was designed to use the antimineralocorticoid property of the riboflavin analogue 7,8-dimethyl-10-(3-chlorobenzyl)isoalloxazine (CBI) to investigate the involvement of mineralocorticoids in the hypertension of the Kyoto strain of the spontaneously hypertensive rat (SHR) and Dahl salt-sensitive (S) rat. Wistar Kyoto (WKY) mildly hypertensive rats were used as controls. The administration of the riboflavin antagonist CBI at 5.0 mg/kg body weight twice weekly for 7 weeks lowered the systolic blood pressure (SBP) of the unanesthetized SHR from 188 +/- 7 mm Hg to 148 +/- 2 mm Hg (P less than 0.05). This was concurrent with a 36% and 11% decrease in iliopsoas muscle Na+ concentration and water content, respectively. The simultaneous administration of CBI and hydrochlorothiazide (HCTZ) reduced the SBP to 126 +/- 4 mm Hg (P less than 0.05). There was a profound suppressive effect of CBI on the secondary hyperaldosteronism generated by HCTZ (17.6 +/- 4.3 vs. 50.4 +/- 7.2 ng/dl, P less than 0.05), which was also reflected in the iliopsoas muscle K+ concentration. The effects of CBI on the SBP and iliopsoas muscle Na+ and K+ concentrations of age-matched WKY mildly hypertensive control rats were qualitatively similar to the effects on the SHR. In contrast to the SHR and the WKY rats, the administration of CBI for 8 weeks at 5.0 mg/kg body weight twice weekly to the Dahl S rats did not reduce their mean SBP (205 +/- 5 vs. 200 +/- 4 mm Hg, not significant). CBI treatment did not significantly decrease iliopsoas muscle Na+ concentration or water content.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of flavokinase (EC 2.7.1.26) by aldosterone in the rat kidney.

The effect of aldosterone on rat renal flavokinase (EC 2.7.1.26) enzymic activity and concentration was investigated in bilaterally adrenalectomized male Sprague-Dawley rats. Flavokinase enzymic activity was measured in the 100,000 X g supernatant of renal cortex and red medulla and was increased after 3 h by 19% and 42%, respectively, as a result of aldosterone (1.5 micrograms/100 g BW) administration. Dual isotope labeling studies revealed increases of 20-30% and 20-35% in the incorporation of [3H]- and [35S]methionine into rat renal cortical and red medullary flavokinase, respectively, as a result of aldosterone administration. The aldosterone-dependent increases in methionine incorporation were blocked when the mineralocorticoid receptor antagonist spirolactone (SC 26304; 150 micrograms/100 BW) was administered 30 min before aldosterone. The relative concentrations of renal flavokinase also increased by 40% in both the cortex and red medulla 3 h after aldosterone treatment, as determined by an enzyme-linked immunosorbent assay (ELISA). These increases were abolished by actinomycin D (100 micrograms/100 g BW) and cycloheximide (200 micrograms/100 g BW), given 1 h before aldosterone administration. The mineralocorticoid receptor antagonists SC 26304 (225 micrograms/100 g BW) and progesterone (500 micrograms/100 g BW) were also able to inhibit the aldosterone-dependent increase in renal flavokinase concentration. The effect of aldosterone on renal flavokinase concentration was studied from 30 min to 8 h after aldosterone administration. There appeared to be maximum increases of 23-30% and 25-32% after 2.5-3.5 h in the renal cortex and red medulla, respectively. 5 alpha-Dihydroaldosterone, a metabolite of aldosterone with mineralocorticoid activity, was also able to increase renal flavokinase concentrations by approximately 40%. However, dexamethasone, a potent glucocorticoid with little or no mineralocorticoid activity, appeared to have no effect on renal flavokinase, as observed by ELISA. These data suggest that the increase in the relative concentration of renal flavokinase may be due to increased biosynthesis of flavokinase, and ultimately, that renal flavokinase may be an aldosterone-induced protein whose synthesis is mediated through the mineralocorticoid receptor and RNA synthesis.

Antihypertensive effect of riboflavin analogs in rats with mineralocorticoid-induced hypertension.

This study investigated whether the riboflavin analogs, 7,8-dimethyl-10-formylmethyl isoalloxazine (FMI) and 7,8-dimethyl-10-(2'-hydroxyethyl) isoalloxazine (HEI), are effective antihypertensive agents in mineralocorticoid-induced or deoxycorticosterone acetate (DOCA)-salt hypertension. These studies are based on our previous observation tht aldosterone enhances the biosynthesis of renal flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) from riboflavin, and that FMI and HEI competitively inhibit conversion of riboflavin to FMN and reabsorption of Na+ in the kidney of adrenalectomized rats. When 1.6 mg of FMI or HEI were administered simultaneously with 3.0 mg of DOCA, the tail systolic blood pressure (SBP) of unanesthetized rats rose only to 136 +/- 5 mm Hg (standard error of the mean, SEM) compared to 163 +/- 5 mm Hg during DOCA therapy alone (p less than 0.0005). This hypotensive effect of FMI or HEI was noted after the fourth week of treatment and persisted through the ninth week. The rats tolerated the medication well and had no signs of riboflavin deficiency. DOCA administration alone resulted in a 24% increase in iliopsoas muscle Na+ concentration (p less than 0.0005), and a 0.8% increase in the water content of the muscle (p less than 0.025), suggesting a positive Na+ balance. Administration of FMI or HEI blunted the ability of DOCA to increase muscle Na+ concentration (p less than 0.025), water content (p less than 0.01). HEI treatment of the Kyoto strain of spontaneously hypertensive rats (SHR) did not lower their mean SBP. Thus it appears that the hypotensive actions of FMI or HEI are closely associated with their ability to modify the effects of mineralocorticoids on NA+ balance.

Aldosterone stimulation of riboflavin incorporation into rat renal flavin coenzymes and the effect of inhibition by riboflavin analogues on sodium reabsorption.

This study was designed to investigate a possible relationship between the effect of aldosterone upon urinary electrolytes and the incorporation of [(14)C]riboflavin into renal [(14)C]flavin mononucleotide (FMN) and [(14)C]flavin adenine dinucleotide (FAD). Adrenalectomized Sprague-Dawley rats that weighed between 185 and 210 g were pretreated with 15 mug/100 g body wt dexamethasone intraperitoneally. 16 h later they were administered aldosterone (1.5 mug/100 g body wt) and [(14)C]riboflavin (5.0 muCi/200 g body wt). The urethra of each rat was ligated, and the rats were sacrificed by decapitation 3 h later. The urine was aspirated from the bladders of each rat and analyzed for total Na(+) and K(+) excretion while the kidneys were removed and the formation of [(14)C]FMN and [(14)C]FAD was determined for each kidney. There was a significant increase in the formation of renal [(14)C]FMN and [(14)C]FAD (27.3 and 14.4%, respectively) after aldosterone treatment. Aldosterone significantly decreased the excretion of Na(+) by 50%, and increased that of K(+) by 55%. To determine if the increased incorporation of [(14)C]riboflavin into renal [(14)C]FMN and [(14)C]FAD was an important intermediary step in the aldosterone-induced alterations in urinary Na(+) and K(+), the riboflavin analogues 7,8-dimethyl-10-formylmethyl isoalloxazine or 7,8-dimethyl-10-(2'-hydroxyethyl) isoalloxazine were given to the animals i.p. to diminish the conversion of riboflavin to FMN by competitively inhibiting the enzyme flavokinase (EC 2.7.1.26). These analogues were found to significantly counteract the decreased urinary excretion of Na(+) as a result of aldosterone from 26+/-9% to 124+/-58% (SEM) with a dose-related response when administered from 10 to 25 mug/100 g body wt. The same doses of the analogues that significantly increased the urinary output of Na(+) when administered simultaneously with aldosterone also significantly decreased the formation of renal [(14)C]FMN from 15+/-4 to 38+/-3% when compared with the effects of aldosterone alone. The analogues exerted no significant effect on the increased urinary excretion of K(+) by aldosterone. The analogues alone had no influence on urinary Na(+) and K(+) output or the formation of renal [(14)C]FMN and [(14)C]FAD at the dose levels that we investigated. These data strongly suggest that the enhanced synthesis of renal FMN and FAD may be a causative factor in the increased reabsorption of Na(+) as a result of aldosterone; and, consequently, riboflavin analogues may function as a novel class of antimineralocorticoids.