Seto, Shinji MD; Nagao, Shoichi MD; Ozeki, Shin-ichiro MD; Tetsuo, Hirokuni MD; Akahoshi, Masazumi MD; Yano, Katsusuke MD
It has been shown that nitric oxide (NO) is synthesized from L-arginine in the central nervous system1 and NO synthase (NOS) activity has been located in neurons within many areas of the brain, including cerebral cortex, striatum, cerebellar cortex, olfactory bulb, and hypothalamus.2,3 The presence of NOS in specific brain regions such as the nucleus tractus solitarius and rostral ventrolateral medulla suggested that NO might be involved in the central neural control of blood pressure (BP).2–4 Several recent studies have implicated an important role of neurogenic component in the BP increase produced by inhibition of NO synthesis.5–8
Chronic salt loading up-regulated the expression of neuronal NOS mRNA in the supraoptic nucleus and paraventricular nucleus of the hypothalamus with a concomitant increase in NOS activity in the hypothalamo-hypophysial system,9–11 leading us to consider a possible functional role of NO in osmoregulation, and consequently in regulation of sodium-fluid balance.
These findings suggest that central NO is involved in the central regulation of BP through the regulation of sympathetic nervous system (SNS) activity and sodium balance. Deoxycorticosterone acetate (DOCA)-salt hypertension is related to increased SNS activity and sodium retention.12 Therefore, in the present study, we investigated whether chronic inhibition of central NOS modulate the development of hypertension in DOCA-salt hypertensive rats and, if it dose, whether or not the modulation of this hypertension is mediated through the regulations of SNS activity and/or sodium balance.
MATERIALS AND METHODS
Animals and Surgical Procedures
Seven-week-old male Wistar rats (Charles River Japan, Atsugi, Japan) were used in this study. All surgical procedures were performed with rats under intraperitoneal injection (IP) of sodium pentobarbital (50 mg/kg). A 22-gauge stainless steel cannula was stereotaxically implanted into the lateral cerebral ventricle; 1.0 mm posterior to the bregma, 1.5 mm lateral from the midline, and 4.5 mm deep from the dura. An osmotic minipump (Alza, model 2 ML4, Palo Alto, CA) filled with either NG-monomethyl L-arginine (L-NMMA) or normal saline was then implanted intraperitoneally and connected through Teflon tubing to the cannula for 4 weeks' continuous intracerebroventricular infusion (ICV) as described previously.13 The location of the ventricular cannula was confirmed by staining the lateral ventricle with methylene blue, which was injected just before killing the animal. Forty-eight hours after the cannula implantation (at day 0), rats were unilaterally nephrectomized, and a silicone rubber sheet (Dow Corning 3110 RTV, Midland, MI) containing 100 mg/kg DOCA implanted subcutaneously. The experiments were approved by the Animal Right Committee, Nagasaki University.
Effect of L-NMMA ICV on BP and Sodium Balance
DOCA-salt rats were divided into 3 groups: L-NMMA ICV at either 0.08 mg/kg/d (DOCA-0.08, n=8) or 0.16 mg/kg/d (DOCA-0.16, n=8), and saline ICV (60 μL/d) rats (DOCA-s, n=9). They were housed in individual metabolic cages in a temperature-controlled room at 22 °C with a 12-hour light/dark cycle. All rats received standard laboratory chow containing 0.4% NaCl (MF, Oriental Yeast Co, Tokyo, Japan) and 0.9% saline to drink. The BP was measured twice at week in conscious, prewarmed restrained rats by tail cuff plethysmography, and body weight was recorded on the same day for 3 weeks after DOCA implantation. Daily 24-hour urine samples were collected, and the 24-hour amounts of food and water (0.9% saline) consumed were recorded. The 24-hour sodium excretion was calculated from the urine volume and urinary sodium content, and total sodium intake was calculated from the amount of food and water consumed. Daily sodium balance was estimated as total sodium intake minus urinary sodium excretion.
BP Responses to Ganglionic Block With Hexamethonium IV
At day 22, indwelling polyethylene catheters (PE10 fused to PE50) filled with heparinized saline were inserted into abdominal aorta for recording BP, and into femoral vein for drug injections. The catheters were passed subcutaneously and brought out through the skin over the scapular region as described previously.14 Rats were allowed 48 hours to recover from surgical stress, then, the studies were performed, although the rats were in conscious resting state. The arterial cannula was connected to a Statham P23Db pressure transducer (Gould Inc, San Juan, Puerto Rico) and the direct mean BP was continuously recorded (Nihon Koden WT-647G, Tokyo, Japan). Rats were received hexamethonium bromide (50 mg/kg, IV bolus) after 60 minutes of stabilization, and the changes in BP were observed for another 30 minutes.
At day 25, the blood of the rat was collected in chilled plastic tubes containing 0.5 mL of 3.8% ethylenediaminetetraacetic acid. Plasma renin activity (PRA), plasma norepinephrine (P-NE), and plasma arginine vasopressin concentration (AVP) were measured. P-NE level was estimated by high-performance liquid chromatography with electrochemical detection. PRA and AVP levels were measured by radioimmunoassay. All blood collections were made in the afternoon between 3 and 5 PM.
Effect of L-NMMA IP on BP and Sodium Balance
Continuous IP infusion of L-NMMA at either 0.08 mg/kg/d (n=6) or 0.16 mg/kg/d (n=6) was conducted by using osmotic minipump in DOCA-salt rats and was used as another control. BP and daily sodium balance were measured under the same protocol as described above.
Effect of L-NMMA ICV on BP and Sodium Balance in Normal Rats
Continuous ICV infusion of L-NMMA at either 0.08 mg/kg/d (n=6) or 0.16 mg/kg/d (n=6) and continuous ICV infusion of saline (n=6) were conducted in normal Wistar rats under the same procedure as described in DOCA-salt rats. BP and daily sodium balance were measured under the same protocol as described above.
L-NMMA, DOCA, and hexamethonium bromide were purchased from Sigma Chemical Co (St Louis, MO).
The results were expressed as mean±SEM. The data were subjected to statistical analysis by analysis of variance and Kruskal-Wallis test was used to evaluate the significance between groups. A P<0.05 was considered significant.
Effect of L-NMMA ICV on BP
Figure 1 depicts changes of BP in DOCA-0.08, DOCA-0.16, and DOCA-s rats. BP did not differ among 3 groups until week 3, after which BP in DOCA-0.08 rats became significantly higher than those in DOCA-0.16 and DOCA-s rats (at day 21, 167.4±3.6 vs. 143.3±3.9 and 150.3±3.9 mm Hg, P<0.01). The differences of BP were also verified by the measurement of direct mean BP (DOCA-0.08; 167.5±3.8 vs. DOCA-0.16; 136.3±2.6 and DOCA-s; 144.4±4.3 mm Hg, P<0.01). There was no significant difference in BP between DOCA-0.16 and DOCA-s rats.
BP Responses to Hexamethonium IV
In all 3 groups, mean BP was substantially reduced by ganglionic blockade with hexamethonium to levels that were no longer significantly different (100.3±4.7, 88.7±3.9, and 97.1±4.8 mm Hg, respectively).
Effect of L-NMMA ICV on Sodium Balance
Figure 2 shows daily food and water (0.9% saline) intake in DOCA-0.08, DOCA-0.16, and DOCA-s rats. Daily food intake did not differ among 3 groups throughout the experiment. On the other hand, at week 2 and thereafter, daily amount of saline drinking was significantly less in DOCA-0.16 rats than those in DOCA-0.08 and DOCA-s rats.
Estimated cumulative sodium retention and body weight were depicted in Figure 3. DOCA-0.16 rats showed a significantly less retention of sodium than DOCA-0.08 and DOCA-s rats (at day 21, 47.5±1.1 vs. 61.7±2.5 and 66.0±3.7 mmol, P<0.01). Body weight in DOCA-0.16 rats became smaller than in DOCA-0.08 and DOCA-s rats after week 3 (at day 21, 325±9 vs. 357±8 and 356±8 g, P<0.05). There was no significant difference in PRA, P-NE, or AVP among these 3 groups (Table 1).
Effect of L-NMMA IP on BP and Sodium Balance
Continuous IP infusion of L-NMMA at either 0.08 mg/kg/d or 0.16 mg/kg/d did not show an augmentation of hypertension and/or an attenuation of sodium retention (Table 2).
Effect of L-NMMA ICV on BP and Sodium Balance in Normal Rats
In normal rats, 4 weeks' continuous ICV of L-NMMA at 0.08 mg/kg/d did not show an increase of BP, whereas ICV at 0.16 mg/kg/d heightened BP significantly compared with saline ICV (Table 3). However, there was no significant difference in BP between L-NMMA ICV at 0.08 and 0.16 mg/kg/d. On the other hand, L-NMMA ICV at either 0.08 or 0.16 mg/kg/d did not change the sodium balance in normal rats.
The present study demonstrated that chronic ICV of L-NMMA displayed a dose-dependent dual effect on BP and sodium balance in DOCA-salt hypertensive rats. The lower dose (0.08 mg/kg/d) of L-NMMA ICV augmented the development of hypertension, but did not alter the sodium balance. On the other hand, the higher dose (0.16 mg/kg/d) did not enhance the hypertension, however, it caused a marked reduction of water (0.9% saline) consumption and of estimated cumulative sodium retention. Because either dose of L-NMMA IP did not affect the development of hypertension and sodium balance, these effects of L-NMMA ICV on BP and sodium balance cannot be attributed to the peripheral action of L-NMMA.
The augmentation of hypertension by lower dose L-NMMA ICV was eliminated after ganglionic blockade with hexamethonium. Hexamethonium competes with acetylcholine for the specific receptors on the ganglion cells, and the effect of hexamethonium can be used to measure the neurogenic component in hypertension. Thus, these observations were indicative of sympathetic stimulation by lower dose L-NMMA ICV. Inhibition of central NO formation by centrally administered L-NMMA has been found to activate the SNS.5,6,8 And, it is well known that the SNS is involved in the development of DOCA-salt hypertension.12 Because the lower dose L-NMMA ICV did not elevate BP in our normal rats, it is suggested that either the L-NMMA–induced sympathetic activation is intensified in DOCA-salt hypertension or DOCA-salt–induced sympathetic activation is enhanced by L-NMMA. And this intensification could bring a further increase of BP. However, P-NE levels did not differ between L-NMMA–treated and saline-treated DOCA-salt rats in this study. Little information is available regarding the effect of central NOS inhibition on P-NE level. Although intravenous injections of NOS inhibitor are reported to elicit an increase in central sympathetic outflow,8,15 other studies have indicated no change in plasma catecholamines levels with chronic NO inhibition despite the increase in SNS activity.8,16,17 Accumulating evidences indicate that reactive oxygen species (ROS) have important effects on central neural mechanisms that implicate hypertension. Further, recent studies hint the close link between NO and ROS with the ability of ROS to quench endogenous NO causing an increase in sympathetic nerve activity.18,19 Also, it has been demonstrated that ROS in the brain contribute, at least partially, to the pressor effect of NOS inhibitor ICV through the stimulation of central and peripheral SNS activity.19 These suggest the possible impact of chronic ICV L-NMMA on levels of ROS in the brain. Unfortunately, we did not examine the levels of ROS and/or antioxidants in this study.
Contrary to the lower dose L-NMMA ICV, chronic inhibition of central NO formation by higher dose L-NMMA did not augment the hypertension in DOCA-salt rats, but attenuated saline drinking at week 2 and thereafter compared with those in lower dose L-NMMA-treated and saline-treated DOCA-salt rats. Central NO has been reported to implicate in drinking behavior with conflicting results.20–22 Calapai et al20 demonstrated that L-arginine administered ICV was able to inhibit drinking in water-deprived rats and they concluded that central NO acts as an inhibitory mechanism through the suppression of central action of angiotensin II when thirst is stimulated. On the other hand, central injection of NOS inhibitors, either L-NMMA or L-NAME, attenuated water consumption induced by osmotic stimulation and hypotensive hemorrhage.21,22 However, those studies were conducted under acute conditions and the effects of chronic inhibition of central NO had not been determined. In this study, chronic central injection of L-NMMA provoked an antidipsogenic effect rather than a dipsogenic response in DOCA-salt rats at week 2 and thereafter. Although renin-angiotensin system is suppressed in DOCA-salt hypertensive rats,23 chronic inhibition of NOS activates the renin-angiotensin system in the brain stem.24 Thus, this antidipsogenic reaction for saline may not be through the inhibitory action on central angiotensin II. Also, it has been demonstrated that NOS gene expression in the supraoptic nucleus and paraventricular nucleus of the rat hypothalamus is increased during hyperosmotic stimulation, and further, a neuromodulatory role for NO in the rat hypothalamo-hypophsial system is suggested as a regulator of AVP secretion.3,9,11 These findings are leading us to consider a possible role of AVP on sodium balance in L-NMMA–treated DOCA-salt rats. However, in the present study, plasma AVP levels did not differ between L-NMMA–treated and saline-treated rats. Although we cannot specify the central site(s) modulating drinking behavior after L-NMMA ICV in this study, it may involve circumventricular organs in the anteroventral part of the third ventricle area. Number of neurones expressing NOS increases in paraventricular nucleus after salt loading,10,11 and the lesions of the subfornical organ and nucleus medianus impair the drinking response to osmotic and angiotensin II stimulation.25,26 Moreover, NOS activity of the subfornical organ decreases after L-NMMA ICV in dehydrated rats.21 On the other hand, in normal rats, chronic L-NMMA ICV at doses used in the present study had no effect on drinking behavior. These suggest that the implication of central NO in drinking behavior would be documented in the situation only when thirst was stimulated through the osmotic and/or angiotensin II stimulation.
Present study demonstrated that chronic inhibition of central NO formation by L-NMMA showed a dual effect in DOCA-salt hypertension; lesser inhibition provoked an augmentation of hypertension through the stimulation of SNS, although more potent inhibition brought an antidipsogenic reaction for saline drinking without an augmentation of hypertension. Dose-dependency of the reduced drinking response to central injection of NOS inhibitor was also reported by Liu et al.22 Although, they showed the dose-dependent pressor effect by NOS inhibitor ICV in hypotensive hemorrhagic rats. Again, in normal control rats, L-NMMA ICV did not show a dual effect on BP and drinking behavior. Instead, higher dose ICV elevated BP and did not show an antidipsogenic effect. The attenuation of saline drinking is likely to account for the lower BP in higher dose L-NMMA than in lower dose L-NMMA ICV DOCA-salt rats. The reduction of sodium retention in higher dose L-NMMA ICV may cancel the pressor effect of L-NMMA ICV. However, if that was the case, it would be expected that ganglionic blockade might cause an exaggerated fall in BP in higher dose ICV DOCA-salt rats than in lower dose ICV rats. At the present study, BP was reduced to the same level in 2 groups by ganglionic blockade. Further studies are needed to clarify the mechanisms of the dual effect of central NO.
Chronic ICV of L-NMMA displayed dual effects on BP and sodium balance depend on the doses in DOCA-salt hypertensive rats. The lesser inhibition of central NO augmented the development of hypertension through the stimulation of SNS, but did not alter the sodium balance. On the other hand, the higher inhibition caused a marked reduction of saline consumption and less sodium retention, but did not affect the development of hypertension. Central NO seems to contribute to the regulation of BP in DOCA-salt hypertension through the dual effects on SNS activity and sodium balance.
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