INTRODUCTION

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THREE RECENT STUDIES have demonstrated that intracerebroventricular infusions of benzamil can prevent the onset of mineralocorticoid-induced and Na⁺-dependent hypertension (7, 8, 18). Benzamil, an amiloride analog, is a Na⁺-channel inhibitor (14), and this has been thought to be the mechanism by which it has its antihypertensive effects. However, the extent to which brain parenchymal cells express amiloride-sensitive Na⁺ channels is in question (5, 16, 27), although amiloride-sensitive nonselective cation channels are present at the cerebral endothelium (26), the site of the blood-brain barrier (BBB). Those endothelial channels are thought to be involved in the movement of Na⁺ and K⁺ between the blood and brain (6, 26), and changes in brain K⁺ regulation have been implicated in the genesis of deoxycorticosterone acetate (DOCA)-salt-induced hypertension (13).

This study examines the effects of intracerebroventricular infusion of benzamil on DOCA-salt-induced hypertension and whether intracerebroventricular benzamil alters BBB Na⁺ transport and brain K⁺ homeostasis. Finally, this study examines whether the effects of benzamil are mediated by a Na⁺ channel by comparing the effects of three different amiloride analogs, benzamil, dimethyl amiloride (DMA), and 3,4-dichlorobenzamil (DCB), which have markedly different affinities for amiloride-sensitive Na⁺ channels (14). Amiloride has effects on a number of transport systems, including Na⁺ channels, Na⁺/H⁺ exchange, and Na⁺/Ca²⁺ exchange (14). Benzamil is more selective than amiloride for the Na⁺ channel, but it also inhibits the other systems. By contrast, DMA preferentially inhibits Na⁺/H⁺ exchange, whereas DCB has a higher affinity for the Na⁺/Ca²⁺ exchanger and a lower affinity for the Na⁺ channel than benzamil.

	MATERIALS AND METHODS

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Experimental protocols. All procedures were approved by the University Committee on Use and Care of Animals at the University of Michigan. Two sets of experiments were performed on adult male Sprague-Dawley rats weighing between 275 and 300 g. The first set examined changes in blood pressure in concious DOCA-salt- and sham-treated rats 2 wk postoperative. The rats received intracerebroventricularly either 300 mM mannitol or 300 mM mannitol with 1 mg/ml benzamil for the 2 wk. Six rats were used for each group. At the end of the infusion period, the rats were used to examine cerebral spinal fluid (CSF) K⁺ homeostasis and blood-to-brain ²²Na⁺ transport. In the second set of experiments, the effect of intracerebroventricular infusions of different concentrations (0.1, 0.3, and 1.0 mg/ml) of either benzamil, DMA, or DCB on the blood pressure was examined in conscious DOCA-salt- and sham-treated rats. Five to six rats were used for each dose. Whether the effects of the highest dose (1.0 mg/ml) of DMA and DCB on blood pressure were mediated centrally was investigated by infusing the same dose subcutaneously. Four rats were used in each of these groups.

Animal preparation and blood pressure measurements. Rats were initially trained for tail-cuff systolic blood pressure measurement on at least three separate occasions to establish a baseline blood pressure. Systolic pressures were then measured every 3-4 days during the study.

After training for the blood pressure measurements, animals were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg) intramuscularly. All animals were uninephrectomized via a flank incision with care being taken to preserve the adrenal gland and its circulation. The animals designated as DOCA-salt also received Silastic implants impregnated with DOCA (200 mg/kg body wt), which were placed subcutaneously in the dorsal neck region. These animals were given water that contained 1.0% NaCl and 0.2% KCl. Sham animals received no Silastic implant and had normal drinking water. All animals were housed individually and allowed access to food and water ad libitum.

At the time of the kidney removal and DOCA implantation, the animals were placed into a stereotaxic apparatus, and 28-gauge stainless steel cannulas (Plastics One, Roanoke, VA) were implanted into the lateral ventricle. The cannulas were then attached to Alzet miniosmotic pumps (model 2002, Alza, Palo Alto, CA) with PE-60 tubing. Minipumps contained 300 mM mannitol as the vehicle with either no amiloride analog or 0.1, 0.3, or 1 mg/ml of benzamil or DMA. For the DCB experiments (including vehicle-alone infusions), the minipumps contained either 1 or 2% dimethyl sulfoxide (DMSO) for solubility reasons. The 1% DMSO was used for 0.1 and 0.3 mg/ml solutions, whereas the 2% DMSO was used for 0.3 and 1 mg/ml solutions. The transition from 1 to 2% DMSO did not affect blood pressure (see RESULTS). The pumps delivered 0.5 µl/h and were implanted subcutaneously in the dorsal neck region. The pump rate is about 1/240th of the normal rat CSF secretion rate of 2 µl/min (11).

For the experiments where DOCA-salt-treated rats received subcutaneous rather than intracerebroventricular infusions of the amiloride analogs, the implanted osmotic minipumps were not attached to a lateral ventricle catheter. Instead, the pumps delivered the amiloride analogs subcutaneously (in the dorsal neck region).

Plasma and CSF electrolytes and blood-to-brain ²²Na transport. After blood pressure was monitored for 2 wk, the first set of rats was anesthetized with pentobarbital (50 mg/kg ip), and two femoral artery and one femoral vein catheters were inserted. One femoral artery catheter was used to measure blood gases and plasma ion concentrations. A CSF sample was then taken from the cisterna magna by puncture of the atlantooccipital membrane with a 26-gauge needle. Plasma and CSF Na⁺, K⁺, and Ca²⁺ concentrations were determined by flame photometry (IL943 flame photometer, Instrumentation Laboratories, Lexington, MA) or an ion-selective electrode (GEM Premier, Mallinckrodt, Ann Arbor, MI). Blood gases and pH were determined with a blood gas analyzer (GEM Premier).

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	RESULTS

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Effects of intracerebroventricular benzamil on DOCA-salt-induced hypertension. As expected, DOCA-salt treatment caused a marked increase in blood pressure over 2 wk (Fig. 1). At 2 wk mean systolic blood pressures were elevated by ~50 mmHg compared with controls (P < 0.001). Intracerebroventricular infusion of benzamil (1 mg/ml at 0.5 µl/h) had no effect on blood pressure in the sham-operated rats, but it significantly blunted the increase in blood pressure found in DOCA-salt-treated rats (P < 0.001; Fig. 1).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Changes in mean systolic blood pressure during a 2-wk intracerebroventricular infusion in sham (squares)- and deoxycorticosterone acetate (DOCA)-salt (circles)-treated rats. Infusions were either 300 mM mannitol alone or mannitol + 1 mg/ml benzamil. Values are means ± SE; n = 5-6 rats. *** Significant difference between the DOCA-salt + mannitol group and each of the other groups at P < 0.001 level.  Significant difference between DOCAsalt + benzamil group and the sham + mannitol group at P < 0.05 level. There were no significant differences between sham groups infused with mannitol and benzamil.

At the end of the 2-wk period, there was no significant difference in body weight between sham-operated and DOCA-salt-treated rats with or without benzamil (Table 1). Similarly, there were no significant differences among the four groups in blood PCO₂ or hematocrit. DOCA-salt-treated rats, with or without benzamil, were alkalotic compared with sham-operated rats. There were also some minor variations in PO₂ among the four groups, but these are likely to have no physiological significance.

The K_i values for blood-to-brain and blood-to-CSF ²²Na⁺ transport were not significantly different in sham-operated and DOCA-salt-treated rats (Fig. 2). Intracerebroventricular benzamil (1 mg/ml) also did not significantly affect the K_i.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Influx rate constant (Ki) for 22Na movement from blood to anterior cortex or cerebrospinal fluid (CSF). Measurements were made in sham- and DOCA-salt-treated rats 14 days after onset of treatment. Rats received an intracerebroventricular infusion of either mannitol (300 mM) or mannitol + benzamil (1 mg/kg) for 14 days. Values are means ± SE; n = 5-6 rats. There were no significant differences among the four groups.

Plasma and CSF [Na⁺] were both unaffected by DOCA-salt treatment (2 wk), and intracerebroventricular benzamil did not alter either parameter in sham- or DOCA-salt-treated rats (Fig. 3A). Compared with sham-operated rats, DOCA-salt-treated rats were hypokalemic (Fig. 3B). The degree of hypokalemia was slightly less in the intracerebroventricular benzamil (1 mg/ml) group, but CSF [K⁺] was unaffected.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Plasma and CSF Na+ (A) and K+ (B) concentrations in sham- and DOCA-salt-treated rats after 14 days. Rats received an intracerebroventricular infusion of either mannitol (300 mM) or mannitol + benzamil (1 mg/kg). Values are means ± SE; n = 5-6 rats. *** Significant difference between DOCA-salt-treated rats and their respective control (i.e., DOCA-salt + benzamil vs. sham + benzamil, or DOCA-salt + mannitol vs. sham + mannitol).

Effects of amiloride analogs on DOCA-salt-induced hypertension. At a dose of 1 mg/ml (at 0.5 µl/h), the highest dose used, none of the three amiloride analogs given intracerebroventricularly had an effect on blood pressure in sham-operated rats after 2 wk (Fig. 4). In contrast, at that dose, all three analogs (benzamil, DMA, and DCB) resulted in a marked reduction in the blood pressure attained after 2 wk in DOCA-salt-treated rats (Fig. 4). The degree of reduction did not vary among the three analogs. At a lower dose (0.3 mg/ml), only DCB (P < 0.01) and benzamil (P < 0.05) significantly attenuated the rise in blood pressure resulting from DOCA-salt treatment (Fig. 4). At the lowest dose used (0.1 mg/ml at 0.5 µl/h), none of the amiloride analogs significantly inhibited the increase in blood pressure induced by DOCA-salt treatment.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Mean systolic blood pressure in sham-operated and DOCA-salt-treated rats receiving intracerebroventricular infusions (0.5 µl/h) of either vehicle or one of three amiloride analogs, benzamil, dimethyl amiloride (DMA), and 3,4-dichlorobenzamil (DCB). Values are means ± SE; n = 5-6 except for vehicle-infused groups where n = 16 and 23 for sham-operated and DOCA-salt-treated rats, respectively, and for 0.3 mg/ml 3,4-dichlorobenzamil group where n = 10. For the latter group, two groups of experiments were performed; one where the minipumps contained 1% DMSO and the other where they contained 2% DMSO. Results from two groups were not significantly different, and experiments were combined. * and ** Significant differences between vehicle- and amiloride-treated DOCA-salt groups at P < 0.05 and P < 0.01 levels, respectively (Dunnett's test).

Subcutaneous infusion of 1 mg/ml (at 0.5 µl/ h) of DMA or DCB failed to affect blood pressure in DOCA-salt-treated rats. Thus, after 2 wk, the blood pressure in DOCA-salt-treated rats receiving 1 mg/ml of DMA was 174 ± 16 mmHg, in rats receiving 1 mg/ml of DCB was 169 ± 11 mmHg, and in rats receiving no amiloride analog was 161 ± 4 mmHg. There were no significant differences among the three groups.

Table 2 displays measurements of plasma and CSF ion concentrations after 2 wk of intracerebroventricular infusion of the highest dose of the amiloride analogs (1 mg/ml) compared with a vehicle infusion. Neither DMA nor DCB affected blood or CSF ion concentrations in sham-operated rats. Similarly, neither of these analogs affected ion concentrations in DOCA-salt-treated rats apart from a slight enhancement of the alkalosis generated by DOCA-salt treatment following intracerebroventricular infusion of DMA.

	DISCUSSION

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This study confirms the effect of intracerebroventricular benzamil on DOCA-salt-induced hypertension. However, this study found no evidence that intracerebroventricular benzamil affected BBB Na⁺ transport or brain K⁺ homeostasis, suggesting that it does not act by affecting the known amiloride-sensitive cation channel at the BBB. Although this result does not indicate whether intracerebroventricular benzamil might inhibit a parenchymal cell Na⁺ channel, a comparison of the effects of different amiloride analogs with those of benzamil suggests that the antihypertensive effects are not mediated by inhibiting such a channel.

Effects of intracerebroventricular benzamil on DOCA-salt-induced hypertension. Gomez-Sanchez and Gomez-Sanchez (7, 8) first reported that intracerebroventricular infusions of benzamil could prevent mineralocorticoid-induced hypertension and salt-induced hypertension in Dahl salt-sensitive rats. Nishimura et al. (18) then expanded this work, demonstrating that intracerebroventricular benzamil could reduce blood pressure in a number of forms of Na⁺-induced hypertension, including DOCA-salt-induced hypertension. These blood pressure effects of benzamil were mediated centrally because systemic administration at the doses used had no effect on blood pressure (7, 8, 18).

In the current study, we have also shown that intracerebroventricular benzamil can prevent the effects of DOCA-salt treatment on blood pressure. These results complement those of Nishimura et al. (18), who found that benzamil could reduce blood pressure in DOCA-salt-treated rats with established hypertension. The doses of benzamil that were effective in our study (0.3 and 1 mg/ml infused at 0.5 µl/h) were the same as the doses used by Gomez-Sanchez and Gomez-Sanchez in their studies (7, 8). The method used in this study to measure conscious systolic blood pressure (the tail cuff) can have limitations in studies where blood pressure changes are small. However, the effect of DOCA-salt treatment on blood pressure and the effect of amiloride analogs on that change are of a considerable magnitude (30-50 mmHg at 14 days), negating the need for measurements with indwelling catheters.

We next examined whether intracerebroventricular benzamil at these concentrations could affect the known amiloride-sensitive cation channel at the BBB (26). This channel is a nonselective cation channel (26), and although it appears to be involved in the movement of Na⁺ from blood to the brain (6), it may also be involved in K⁺ transport at the BBB (12). Infusing benzamil at 1 mg/ml at 0.5 µl/h failed to alter either blood-to-brain ²²Na transport or CSF K⁺ concentration, even though it did ameliorate the effects of DOCA-salt treatment on blood pressure, indicating that these antihypertensive effects are not mediated by the inhibition of the BBB ion channel.

In addition, although intracerebroventricular infusions of hypertonic NaCl (1, 25) induce increases in blood pressure, and changes in CSF Na⁺ concentration have been implicated in the genesis of Na⁺-dependent hypertension (17), we found no effect of benzamil (or any of the amiloride analogs) on CSF Na⁺. Indeed, in common with the findings of Takata et al. (23) and Nishimura et al. (18), we found no effect of DOCA-salt treatment on CSF Na⁺ concentration.

Effects of amiloride analogs. The absence of an effect of benzamil on BBB Na⁺ transport (and CSF Na⁺ and K⁺ concentration) suggests that benzamil does not have its antihypertensive effect by inhibiting the known BBB nonselective cation channel. A comparison of the effects of the different amiloride analogs on DOCA-salt-induced hypertension suggests that the antihypertensive effects are not mediated by the inhibition of a parenchymal cell Na⁺ channel but may rather be via an alteration in brain Ca²⁺ homeostasis. As with benzamil (7, 8, 18), the blood pressure effects of DMA and DCB appear to be mediated centrally because systemic administration did not affect blood pressure in DOCA-salt-treated rats.

Amiloride has effects on a wide range of ion transporters and ion channels, including Na⁺/H⁺ exchange and certain Na⁺ channels (reviewed in Ref. 14). Specific amiloride analogs have been developed that have different affinities for these transporters. Thus benzamil inhibits amiloride-sensitive Na⁺ channels at concentrations 10 times lower than with amiloride and 100 times lower than with DMA (14). In contrast, DMA inhibits Na⁺/H⁺ exchange at concentrations 10 times lower than with amiloride and 100 times lower than with benzamil (14). The fact that benzamil and DMA have potencies for the Na⁺ channel and the Na⁺/H⁺ exchanger that differ by two orders of magnitude but have almost the same potency in inhibiting the hypertensive effect of DOCA-salt treatment suggests that this antihypertensive effect is not mediated through a Na⁺ channel or Na⁺/H⁺ exchange.

Amiloride and its analogs are also inhibitors of Na⁺-K⁺-ATPase and Na⁺-coupled transport (e.g., Na⁺-PO³₄ transport). However, these actions are unlikely to be involved in the antihypertensive actions described in this study. Inhibition of these types of transport requires concentrations close to the millimolar range (14), and the concentration in CSF in the present experiments may, at the highest dose, be closer to 10-15 µM [rat CSF production of 2 µl/min (11) would be expected to dilute the infusate 240-fold]. In addition, central administration of a Na⁺-K⁺-ATPase inhibitor ouabain actually causes increases in blood pressure (9).

Amiloride analogs also inhibit Na⁺/Ca²⁺ exchange (14). Unlike potencies for the Na⁺ channel and the Na⁺/H⁺ exchanger, the potencies of DMA and benzamil for the Na⁺/Ca²⁺ exchanger differ by less than an order of magnitude with benzamil having the higher affinity. DCB has a higher affinity for the Na⁺/Ca²⁺ exchanger than either of the other amiloride analogs, but its potency is still only about 10 times that of DMA. Thus, unlike with the Na⁺ channel and the Na⁺/H⁺ exchanger, the relative potency of the different amiloride analogs for inhibiting Na⁺/Ca²⁺ exchange fairly closely matches their antihypertensive effects. In vitro with astrocytes and brain membrane preparations, DCB inhibits Na⁺/Ca²⁺ exchange with an IC₅₀ of about 15-30 µM (10, 24). These values are similar to the CSF concentrations expected from the intracerebroventricular infusions used in the experiments in this study.

There have been numerous studies in humans and animals examining the role of Ca²⁺ in the regulation of blood pressure (reviewed in Ref. 21) and intracerebroventricular infusions of Ca²⁺ lower blood pressure (4, 15, 22). Amiloride analog-induced inhibition of Na⁺/Ca²⁺ exchange might be expected to have a similar effect on blood pressure by raising intracellular Ca²⁺ concentrations. Amiloride analogs can also inhibit Ca²⁺ channels (3, 14), but such inhibition would be expected to lower intracellular Ca²⁺ concentrations.

In summary, these results support previous findings on the antihypertensive effects of intracerebroventricular benzamil but suggest that these are not mediated by the inhibition of brain Na⁺ channels. Rather, the intracerebroventricular administration of amiloride analogs may ameliorate the effects of DOCA-salt treatment on blood pressure by inhibiting Na⁺/Ca²⁺ exchange.