l-Arginine Prevents Corticotropin-Induced Increases in Blood Pressure in the Rat
Abstract In this study we examined whether l-arginine treatment could prevent corticotropin (ACTH)–induced increases in blood pressure in the Sprague-Dawley rat. Sixty rats were randomly divided into six groups (n=10): sham injection, ACTH injection (0.5 mg/kg per day in divided doses), l-arginine (0.6%) in food plus sham injection, l-arginine plus ACTH treatment, d-arginine (0.6%) in food plus sham injection, and d-arginine plus ACTH. Systolic pressure, water intake, urine volume, body weight, plasma and urinary electrolytes, and serum corticosterone concentrations were measured. ACTH increased systolic pressure (from 127±2 to 165±6 mm Hg, P<.001), water intake, and urine volume and decreased body weight. l-Arginine reduced ACTH-induced blood pressure rises (130±3 mm Hg, P<.001) but had no effect on blood pressure in sham-treated rats. d-Arginine did not affect blood pressure in sham-treated rats, and systolic pressure in d-arginine+ACTH–treated rats was similar to that of ACTH-treated rats. l-Arginine decreased serum corticosterone concentrations in sham-treated rats (424±43 versus 238±25 ng/mL, P<.01), but d-arginine had no effect. However, both drugs decreased serum corticosterone concentrations in ACTH-treated rats (1071±117 versus 739±95 and 695±72 ng/mL for l- and d-arginine, respectively; both P<.05). As l-arginine but not d-arginine prevented ACTH-induced increases in blood pressure in Sprague-Dawley rats and both l- and d-arginine reduced serum corticosterone concentrations in ACTH-treated rats, the effects of l-arginine in preventing ACTH-induced hypertension were not simply a consequence of decreased corticosterone secretion.
In 1987, NO was identified as an endothelium-derived relaxing factor.1 2 NO is synthesized from L-Arg by a family of enzymes, the NO synthases, and is spontaneously oxidized to nitrite and nitrate with a half-life of 6 to 10 seconds.3 The NO–L-Arg pathway plays an important role in BP regulation.4 5 6 7
ACTH injection raises BP in rats,8 9 10 and the rise depends on intact adrenal glands.9 Inhibition of NO synthesis with oral N-nitro-l-arginine raises BP in SD rats and has an additive effect on ACTH-induced hypertension.11 These results are compatible with the notion that the hypertension induced by ACTH is not a consequence of NO inhibition but could also be explained by partial inhibition of NO by both treatments. Glucocorticoids are known to inhibit the expression of an inducible NO synthase in vascular endothelial cells but are reported not to affect the constitutive enzyme.12 L-NAME, an NO synthase inhibitor, increased corticosterone secretion dose dependently in the rat, suggesting that adrenal steroidogenesis was negatively regulated by endogenous NO.13
In 1991, Chen and Sanders14 reported that exogenous L-Arg decreased BP to normotensive levels in Dahl salt-sensitive rats but did not modify BP in salt-resistant rats. In contrast, L-Arg did not alter the development of hypertension in spontaneously hypertensive rats.14 The present study examined the effects of D- and L-Arg on ACTH-induced increases in BP and serum corticosterone concentration in SD rats.
This study was approved by the Animal Care and Ethics Committee of the University of New South Wales (ACE 93/117). Sixty male SD rats (body weight, approximately 200 g) were housed in plastic cages (four rats per cage) at a constant temperature (21° to 23°C). They were fed a commercial diet (Gordon’s Specialty Stock Feeds Pty Ltd) that contained 1.53% arginine and had free access to tap water ad libitum. Twenty-four-hour food and water intakes, urine volume, and body weight were measured every second day with rats in metabolic cages. Twenty-four-hour urine samples were collected for electrolyte determination. Control measurements were performed for 6 days, followed by 10 days of sham or ACTH treatment. At the end of the experiment, all rats were euthanatized under pentobarbital sodium anesthesia (60 mg/kg IP). The time from anesthesia to sampling was similar in all groups. Blood samples (6 to 8 mL) were collected from the cannulated carotid artery with rats under pentobarbital sodium anesthesia (60 mg/kg) and stored at −20°C for later assay after centrifugation at 3500 rpm for 15 minutes. A polyethylene catheter (OD of 0.94 mm and ID of 0.75 mm, Portex, Boots Pty Ltd) was used for the cannulation via a midline neck incision.
Rats were randomly divided into six groups (n=10 for each). Group 1 (sham treatment) rats received 0.9% NaCl (0.25 mL/kg SC) twice daily at 10 am and 6 pm for 10 days (T1 through T10). Group 2 (ACTH treatment) rats were injected subcutaneously twice daily at 10 am and 6 pm with synthetic ACTH (0.5 mg/kg per day, 0.25 mL/kg) (Synacthen Depot, CIBA-Geigy) for 10 days (T1 through T10). Group 3 (L-Arg+sham treatment) rats were fed 50 g of food containing 0.6% L-Arg daily at 10:30 am and received 0.9% NaCl by injection as for group 1. The food was made by mixing 24 g of L-Arg (free base) powder (Sigma Chemical Co) with a commercial food powder (4000 g, Gordon’s Specialty Stock Feeds). Group 4 (L-Arg+ACTH treatment) rats were fed with L-Arg as for group 3 and injected with ACTH as for group 2. Group 5 (D-Arg+sham treatment) rats were fed as for group 3 with D-Arg (free base) (Sigma) instead of L-Arg. Group 6 (D-Arg+ACTH treatment) rats were fed D-Arg as for group 5 and injected with ACTH as for group 2.
SBP was recorded on alternate days by the tail-cuff method (Narco Biosystems, Inc) in conscious rats. At least four consecutive cycles (inflation/deflation) were performed on each rat, and the mean of the last three recordings, among which there was not more than 10 mm Hg difference, was taken as the SBP. The recordings taken over the 6 control days, were used as baseline values.
Organ Weight Measurement
At death, adrenal, kidney, and heart weights were measured and expressed relative to body weight (grams organ weight per 100 g body weight).
All urine, plasma, and serum samples were stored at −20°C until analysis. Plasma and urine samples were centrifuged at 3000 rpm for 10 minutes and diluted 1:5 with distilled water; sodium and potassium concentrations were measured by flame photometry (model I.L 943 flame photometer, Coulter Pty Ltd).
Serum Corticosterone Measurement
Serum samples were collected in refrigerated plain Vacutainer tubes 2.5 hours after ACTH injection and stored at −20°C after centrifugation (3500 rpm for 15 minutes). Serum corticosterone was measured by radioimmunoassay (DPC Coat-A-Count rat corticosterone kit). The sensitivity of the assay is approximately 5.7 ng/mL, and the interassay and intra-assay variations were 4.8% and 4%, respectively, at mean values of 421 and 427 ng/mL.
Results are expressed as mean±SEM and were analyzed by two-way repeated measures ANOVA for comparison of all groups together and individual treatment groups with the appropriate control group as well as for analysis of a single treatment group over time. Pretreatment days were pooled to give a mean pooled control. Student’s t tests were performed to compare the group mean of control days with each treatment day and the data between groups. The probability value for Student’s t test was modified by the Hochberg method of multiple comparisons.
The Table⇓ shows plasma electrolyte concentrations, organ weights, and body weights at death in the various rat groups.
Group 1: Sham Treatment
Sham treatment did not change SBP or urinary Na+ or K+ excretion but was associated with increased body weight, urine volume, water intake, and food intake (Figs 1⇓ and 2⇓). Food intake was 28±1 g (pooled control) and ranged from 29±1 to 32±1 g during treatment. Serum corticosterone concentration at death was 424±43 ng/mL (Fig 3⇓).
Group 2: ACTH Treatment
ACTH treatment increased SBP (from 127±2 to 165±6 mm Hg, P<.001) and also reduced body weight and increased water intake and urine volume (Figs 1⇑ and 2⇑). Food intake was 28±1 g (pooled control) and ranged from 24±1 to 32±1 g during treatment. Urinary Na+ excretion increased on days T1, T5, and T7, with no change in urinary K+ excretion (Fig 2⇑). Serum corticosterone concentration at death was 1071±117 ng/mL (Fig 3⇑).
Group 3: L-Arg+Sham Treatment
SBP, urine volume, and food intake did not change during L-Arg treatment, whereas body weight increased and water intake decreased on days T1 and T3 (Figs 1⇑ and 4⇓). Food intake was 29±1 g (pooled control) and ranged from 29±1 to 32±2 g during treatment. L-Arg ingested varied from 612±25 (T1) to 616±18 (T9) mg/kg per day. Urinary Na+ excretion decreased only on day T5, and urinary K+ excretion consistently decreased (Fig 4⇓). Serum corticosterone concentration at death was 238±25 ng/mL (Fig 3⇑).
Group 4: L-Arg+ACTH Treatment
SBP was unchanged during L-Arg+ACTH treatment, whereas body weight decreased, and urine volume and water intake increased (Figs 1⇑ and 4⇑). Food intake was 27±1 g (pooled control) and ranged from 24±2 to 33±1 g during treatment. L-Arg ingested varied from 746±22 (T1) to 669±27 (T9) mg/kg per day. Urinary Na+ and K+ excretions remained unchanged (Fig 4⇑). Serum corticosterone concentration at death was 739±95 ng/mL (Fig 3⇑).
Group 5: D-Arg+Sham Treatment
SBP, urine volume, urinary Na+ excretion, and food intake did not change significantly during D-Arg treatment, but body weight increased and water intake decreased on day T3 (Figs 1⇑ and 5⇓). Food intake was 28±1 g (pooled control) and ranged from 25±0 to 29±1 g during treatment. D-Arg ingested varied from 554±74 (T1) to 582±18 (T9) mg/kg per day. Urinary K+ excretion increased from day T5 on (Fig 5⇓). Serum corticosterone concentration at death was 393±23 ng/mL (Fig 3⇑).
Group 6: D-Arg+ACTH Treatment
SBP increased (from 124±1 to 152±5 mm Hg, P<.001) during D-Arg+ACTH treatment; body weight decreased, and urine volume and water intake increased (Figs 1⇑ and 5⇑). Food intake was 30±1 g (pooled control) and ranged from 25±0 to 34±2 g during treatment. D-Arg ingested varied from 763±39 (T1) to 679±43 (T9) mg/kg per day. Urinary Na+ excretion increased on days T5 and T7, as did urinary K+ excretion on days T3 through T9 (Fig 5⇑). Serum corticosterone concentration at death was 695±72 ng/mL (Fig 3⇑).
Group 1 (Sham Treatment) With Group 2 (ACTH Treatment)
SBP was consistently higher during ACTH than sham injection (Figs 1⇑ and 2⇑). In rats receiving ACTH, water intake, urine volume, and plasma Na+ concentration were higher and body weight and plasma K+ concentration lower. ACTH produced marked increases in adrenal, kidney, and heart weights and serum corticosterone concentration compared with sham injection.
Group 1 (Sham Treatment) With Group 3 (L-Arg+Sham Treatment)
No differences were observed in SBP or metabolic effects between groups 1 and 3. Plasma and urinary K+ and serum corticosterone concentrations were lower, and heart weight was higher in L-Arg–treated rats.
Group 1 (Sham Treatment) With Group 5 (D-Arg+Sham Treatment)
No significant differences were observed in SBP, urine volume, body weight, kidney weight, urinary Na+ and K+ excretions, or serum corticosterone concentration between groups 1 and 5. Compared with sham-treated rats, D-Arg–treated rats showed decreased water and food intakes and lower plasma Na+ and K+ concentrations and adrenal weights.
Group 2 (ACTH Treatment) With Group 4 (L-Arg+ACTH Treatment)
SBP was lower in rats treated with L-Arg+ACTH compared with rats treated with ACTH alone (Fig 1⇑). Urine volume, food intake, adrenal weight, urinary Na+ excretion, and plasma K+ concentration did not differ significantly. Water intake was lower during L-Arg on days T5 and T7. The L-Arg+ACTH–treated group lost less weight, and urinary K+ excretion was lower in L-Arg–treated rats on days T1, T5, and T7. L-Arg+ACTH–treated rats had higher heart weights and lower plasma Na+ concentrations, kidney weights, and serum corticosterone concentrations.
Group 2 (ACTH Treatment) With Group 6 (D-Arg+ACTH Treatment)
SBP was similar in groups 2 and 6, except for day T6, when it was lower in D-Arg+ACTH–treated rats (Fig 1⇑). Body weight, food intake, adrenal weight, urinary Na+ excretion, and plasma Na+ concentration did not differ. Urine volume was lower in the D-Arg+ACTH group on days T5 and T7 and water intake was lower on days T1 through T9, but urinary K+ excretion was higher in D-Arg–treated rats on days T5, T7, and T9. D-Arg+ACTH–treated rats also had higher heart weights and lower plasma K+ concentrations, kidney weights, and serum corticosterone concentrations.
Group 3 (L-Arg+Sham Treatment) With Group 4 (L-Arg+ACTH Treatment)
SBP was increased in the L-Arg+ACTH group compared with the L-Arg+sham group on days T4 and T10 (Figs 1⇑ and 4⇑). Urine volume was higher in L-Arg+ACTH–treated rats, as was water intake, but body weight was lower. Food intake was initially increased in L-Arg+ACTH–treated rats on day T1 but subsequently became lower than that of L-Arg+sham rats. Similar amounts of L-Arg were ingested. In the L-Arg+ACTH group, adrenal, kidney, and heart weights were higher, as was serum corticosterone concentration, but plasma K+ concentration was lower. Urinary Na+ excretion was increased in L-Arg+ACTH–treated rats on days T3, T5, and T9 as well as urinary K+ excretion on days T3 and T5.
Group 5 (D-Arg+Sham Treatment) With Group 6 (D-Arg+ACTH Treatment)
SBP was higher in the D-Arg+ACTH group (Figs 1⇑ and 5⇑), as was water intake and urine volume. Body weight was lower in D-Arg+ACTH–treated rats, and although food intake was similar, D-Arg+ACTH–treated rats ingested more drug than D-Arg+sham rats. D-Arg+ACTH treatment produced greater adrenal, kidney, and heart weights and higher serum corticosterone and plasma Na+ concentrations. Plasma K+ was lower during ACTH.
Group 6 (D-Arg+ACTH Treatment) With Group 4 (L-Arg+ACTH Treatment)
SBP was higher in D-Arg+ACTH–treated rats (Fig 1⇑). Drug intake, metabolic effects, organ weights, urinary Na+ excretion, and serum corticosterone concentration did not differ significantly between the two groups. Plasma Na+ concentration and urinary K+ excretion were higher in D-Arg+ACTH–treated rats.
Although the participation of the L-Arg–NO pathway in the pathogenesis of hypertension has been extensively studied, the role of NO deficiency in hypertension has not been fully defined. L-Arg is a specific precursor of NO synthesis7 and can reverse the hypertension induced by competitive inhibition of NO synthase (eg, NG-monomethyl-l-arginine, L-NAME, and N-nitro-l-arginine).5 7 15 16 L-Arg prevented the development of salt-sensitive hypertension in Dahl/Rapp rats but did not alter the BP rise of spontaneously hypertensive rats.14 Kirchner et al17 reported that L-NAME produced smaller increases in mean arterial pressure in anesthetized DOCA-salt rats than in sham rats, but acute L-Arg administration failed to reduce pressure in anesthetized DOCA-salt rats. Chronic oral and intraperitoneal L-Arg administration did not lower pressure in conscious DOCA-salt rats with established hypertension, nor did it prevent hypertension when given to prehypertensive DOCA-salt rats.17 These observations indicated that the BP lowering of L-Arg seen in the Dahl salt-sensitive rat is a relatively specific effect.
L-Arg infusion induced short-lasting hypotension in both normotensive and essential hypertensive men and in normotensive rats.18 19 20 In humans, L-Arg infused into the left cubital vein over 30 minutes caused rapid-onset decreases in both SBP and diastolic pressure; with cessation of the infusion, BP returned to baseline after 20 minutes.18 However, in high doses both D- and L-Arg have vasodilator effects in the human forearm.21 In conscious unrestricted rats, a bolus of L-Arg produced a marked, dose-related but short-lasting hypotensive effect, which was blocked by pretreatment with atropine.19
We found that oral L-Arg attenuated the rise in BP produced by ACTH. It did not affect BP in control male SD rats, consistent with most previous reports.5 16 The decrease in urinary potassium excretion in the L-Arg+sham group may have been a consequence of a lower plasma potassium concentration, implying that potassium is moving into the cells or being sequestered in the gut, and was unrelated to urine output, food or water intakes, and serum corticosterone concentration. D-Arg did not modify ACTH hypertension (apart from day T6), indicating that the L-Arg effect is stereospecific. The amounts of D- and L-Arg given in our experiment were consistent with those in the previous study by Chen and Sanders,14 and the amounts of drug ingested were similar in all groups, indicating that the SBP and metabolic effects observed were not a consequence of fluctuating food intake.
In 1989, Palacios et al22 reported that incubation of adrenal cytosol with L-Arg stimulated guanylate cyclase activity accompanied by the formation of NO and citrulline, and this effect of L-Arg was inhibited by hemoglobin and NG-monomethyl-l-arginine, demonstrating that rat adrenals contain a constitutive NO synthase in both the cortex and medulla. The findings that L-Arg directly stimulated adrenal NO synthesis in vitro suggested that the L-Arg–NO pathway may participate in the regulation of the secretion or action of glucocorticoids. In 1991, Adams et al13 reported that L-NAME increased corticosterone secretion dose dependently in the SD rat, suggesting that adrenal steroidogenesis was negatively regulated by endogenous NO.
In the present study, oral L-Arg but not D-Arg decreased serum corticosterone in sham-treated rats, consistent with the notion that L-Arg has effects on steroidogenesis attributable to NO.13 22 In contrast, both L- and D-Arg decreased serum corticosterone in ACTH-treated rats. This decrease could reflect increases in hepatic blood flow and hence corticosterone clearance, as at high doses both drugs are vasoactive.21 However, serum corticosterone concentrations in the L-Arg+ACTH–treated rats were still markedly increased compared with control rats and similar to those in D-Arg+ACTH–treated rats. Thus, the effect of L-Arg in preventing ACTH-induced hypertension is unlikely to reflect the blunting of ACTH-stimulated corticosterone secretion. However, the observations do raise the possibility that the role of L-Arg in other forms of hypertension, such as Dahl salt-sensitive hypertension, may in part reflect decreasing steroid production. In the Dahl/Rapp rat, oral L-Arg completely prevented salt-sensitive hypertension.13 This inhibition of salt-sensitive hypertension by L-Arg was prevented by dexamethasone,23 suggesting a role for dexamethasone-suppressible NO synthesis in the prevention of the hypertension. However, as L-Arg decreases steroid production, the role of dexamethasone might be permissive.
It has been assumed in a number of studies that oral L-Arg modulates BP by increasing vasodilator NO synthesis.14 23 24 Several reports indicated that there might be some impairment of NO synthesis in some varieties of hypertension.14 25 26 There are at least two types of NO synthase.7 One is constitutive and Ca2+/calmodulin dependent and releases NO for short periods in response to receptor or physiological stimulation.7 The other enzyme is inducible and Ca2+ independent and synthesizes NO for long periods.7 It is induced by endotoxin and some cytokines, and this induction is inhibited by glucocorticoids.7 12 27 Given that the effects of L-Arg in preventing and reducing ACTH hypertension in the rat do not appear to be a consequence of metabolic effects and are not simply explained by decreases in serum corticosterone concentrations, steroid-induced inhibition of NO activity leading to loss of vasodilatation is a likely candidate mechanism, but we did not examine this proposition in the present study.
In summary, oral L-Arg but not D-Arg decreased ACTH-induced increases in BP in SD rats. L-Arg decreased serum corticosterone concentrations in sham rats, and both L- and D-Arg reduced serum corticosterone in ACTH-treated rats. The effects of L-Arg on ACTH-induced hypertension cannot be attributed simply to changes in ACTH-stimulated corticosterone production.
Selected Abbreviations and Acronyms
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|SBP||=||systolic blood pressure|
This work was supported by the Clive and Vera Ramaciotti Foundation, a General Development Grant from the University of New South Wales, and the National Health and Medical Research Council of Australia. ACTH was generously provided by CIBA-Geigy Australia Ltd. We thank Stephen Ireland, Ross Allen, and Dianne Degouveia of Southpath Division of Chemical Pathology, the St George Hospital, for electrolyte analyses.
- Received June 27, 1995.
- Revision received August 29, 1995.
- Accepted October 24, 1995.
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