Introduction

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The primary involvement of the kidney in the development of some forms of human essential and animal genetic hypertension has been clearly demonstrated (Ferrari et al., 1995; Woolfson et al., 1996). Studies in the Milan-hypertensive (MHS) and -normotensive (MNS) strains of rats (Bianchi et al., 1994) and in essential hypertensive patients (Casari et al., 1995; Cusi et al., 1997) have shown that mutations in the genes coding for the cytoskeletal protein adducin are genetically associated with hypertension and salt sensitivity. In MHS rats adducin mutations are linked to increased expression and maximal activity of the renal Na-K pump (Ferrandi et al., 1996), which is the most important mechanism regulating constitutive tubular sodium reabsorption in the kidney. Moreover, elevated levels of an endogenous inhibitor of the Na-K pump, the so-called ouabain-like factor (OLF), have been observed in adult MHS rats (Ferrandi et al., 1992, 1997a). The sequence of mechanisms that link adducin mutations to the up-regulation of renal Na-K pump expression and activity, enhanced tubular sodium reabsorption and increased OLF levels in MHS have not yet been fully elucidated. According to the mechanism well established for high ouabain concentrations, increased OLF levels should result in an inhibition rather than a stimulation of the renal Na-K pump activity (Smith, 1988). However, low concentrations of ouabain can stimulate Na-K pump activity in cardiac tissue (Ghysel-Burton and Godfraind, 1979; Godfraind et al., 1983; Ghysel-Burton et al., 1983); and long-term treatment of cultured cells with low K⁺, or low concentrations of ouabain (Pollack et al., 1981; Tang and McDonough, 1992), or in vivo chronic digitalization (Wai Ching Li et al., 1993) produce an up-regulation of the Na-K pump units, measured either as activity at V_max or protein expression. This can be considered a long-term positive feedback mechanism by which the cell can re-establish the equilibrium of the Na-K ion gradient altered by pump inhibition (Rayson and Gupta, 1985). In keeping with these findings, it has recently been demonstrated that a common feature of both MHS genetic hypertension and experimental hypertension induced in the rat by chronic infusions of low doses of ouabain (used as an OLF analog) is an increased activity at V_max of the basolateral renal Na,K-ATPase (Ferrari et al., 1998). Therefore, the normalization of both renal Na,K-ATPase up-regulation and OLF effects (or levels) may be targets for the treatment of hypertension in MHS rats.

Our group has recently demonstrated that a new digitoxigenin derivative, PST 2238, able to displace ouabain from Na,K-ATPase in vitro and devoid of any activity on receptors involved in blood pressure and hormonal regulation, antagonizes the pressor effect of ouabain in vivo and normalizes ouabain-dependent hyperactivation of the Na-K pump both in cultured cells and in vivo (Ferrari et al., 1998). PST 2238 has therefore been proposed as a prototype of a new class of antihypertensive compounds able to correct the alterations of renal Na,K-ATPase induced by ouabain or associated with increased OLF. On this basis, we investigated the use of PST 2238 in the treatment of genetic forms of hypertension, such as that of MHS rats, in which these alterations are present. We found that PST 2238 prevented the development of hypertension and normalized renal Na,K-ATPase activity and expression in MHS rats when given orally at very low doses. Moreover, PST 2238 reduced the Na-K pump hyperactivation induced in cultured rat renal cells by transfection with the hypertensive adducin variant but did not affect the pump in the wild-type cell line.

	Materials and Methods

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Animals

Young (25-27 days of age) and adult (60 days of age) MHS/Gib-hypertensive and MNS/Gib-normotensive rats were obtained from the internal stock colony (Prassis-Sigma Tau, Settimo M.se, Milan, Italy). Young (25-27 days of age) male spontaneously hypertensive rats, Okamoto strain (SHR), were purchased from Charles River (Calco, Como, Italy). The rats were housed in groups of five or three, according to age, with a 12-h light/dark cycle at a temperature of 22 ± 2°C, relative humidity of 55 ± 15%, and free access to food (Altromin MT-Rieper, Vandois, Italy) and water. All animal use procedures were in accordance with the European Community Directive 8/609/CEE of November 24, 1986 and relative Italian Law DL n.116 of January 27, 1992.

Materials

The synthesis of PST 2238 has been reported elsewhere (Quadri et al., 1997).

K-canrenoate was purchased from Sigma (St. Louis, MO). For in vivo treatments, both PST 2238 and K-canrenoate were administered orally, by gavage, in suspension with 0.5% v/v methylcellulose (Methocel) at the volume of 100 µl/100 g b.wt.

Experimental Design

Long-Term Treatment of MHS, MNS, and SHR Rats. A first series of experiments examined the effects of PST 2238 on age-dependent blood pressure increase, heart rate (HR), and renal Na,K-ATPase activity and expression in MHS and MNS rats treated for 5 to 6 weeks starting from the prehypertensive age. MHS rats develop hypertension at weaning (3-4 weeks of age) and achieve a stable hypertension at approximately 2 months of age, whereas MNS remain normotensive throughout life (Barber et al., 1994). Before starting the experiments, young rats were accustomed to handling by the same researcher to reduce the influence of stress on the first blood pressure recording. Rats were divided into groups that received PST 2238 at different doses or the vehicle alone (methylcellulose, 0.5% w/v). The compound or vehicle was administered by oral gavage in a volume of 2 ml/kg b.wt. Systolic blood pressure (SBP) and HR were measured weekly at the tail by plethysmography (BP Recorder, U. Basile, Italy) in conscious rats. In one experiment, BP was also measured directly at the carotid: arterial polyethylene catheters (PE₅₀; #Intramedic Clay Adams, NJ) were surgically implanted in rats under halothane anesthesia. Four to 5 h after surgery, when the animals were completely conscious and freely moving, the arterial catheter was connected to a Statam-Gould P23XL pressure transducer (Oxnard, CA) for BP measurement. BP and HR were recorded continuously on a polygraph (Gould two-channels, RS 3200) for 1 h, starting 6 h after the last treatment, and the values for SBP, metabolic blood pressure (MBP), and HR were read approximately every 5 min, taking care that the animals was quiet. The mean of all readings was taken as final value for SBP, MBP, and HR.

Short-Term Treatment of MHS Rats. This experiment was performed to measure the effects of a short-term (10 days) treatment with PST 2238 on SBP and HR in adult MHS rats in which hypertension is fully stabilized. Rats were divided into groups of 5 or 10 animals that received PST 2238, at different doses (0.1, 1, and 10 mg/kg per os), or the vehicle (methylcelluose, 0.5% w/v). The compound or vehicle was administered by oral gavage in a volume of 2 ml/kg b.wt. SBP and HR were recorded daily at the tail of conscious rats as described above and continued to be recorded for a washout period of 2 to 6 days after the end of treatment.

Cell Culture Studies

Cell Culture and Transfection. NRK-52E cells (epithelial-like cells) (De Larco et al., 1978) were purchased from the European Collection of Animal Cell Cultures (CRL 1571). This cell line was selected because its adducin genotype (FR) allowed the reconstitution of the full "hypertensive" double-mutated adducin by simply transfecting it with mutated -adducin cDNA (Tripodi et al., 1996).

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Cell Na-K Pump at V_max. The filters were washed in K⁺-free saline and incubated without external K⁺ for 50 min to load the cells with Na⁺ and reach maximal Na-K pump activation. Na-K pump activity was measured in duplicate as ouabain-sensitive ⁸⁶Rb⁺ uptake (8 µCi/ml) during the initial 10 min from restoration of normal external K⁺ concentration (5.4 mM) as described by Bowen et al. (1992). Ouabain at maximal concentration (10 mM) was added on the basolateral side 5 min before the assay. In the treated cells, PST 2238 was present throughout sodium loading and ⁸⁶Rb uptake. At the end of the uptake period, the filters were washed three times in 250 ml of saline. Radioactivity was extracted by lysing the cells with 0.1 N NaOH and 0.1% sodium dodecyl sulfate (SDS) and counted in a gamma counter (Beckman 5500). An aliquot of the lysate was used to measure the protein content of each filter (Lowry et al., 1951). Ouabain-sensitive Rb⁺ uptake was expressed as the equivalent K⁺ transport in nanomoles per hour per milligram of protein. It was also expressed as rate constant for Na⁺ (h¹), correcting the expected value of Na⁺ extrusion with the intracellular Na⁺ content (see below).

Intracellular Na⁺ Content at V_max. A different set of filters, treated as described above, were used to measure intracellular sodium content after loading. The filters were washed four times in an Na⁺-free medium: 95 mM choline chloride, 1 mM MgCl₂ , 85 mM sucrose, 10 mM glucose, and 10 mM 4-morpholinepropanesulfonic acid-Tris buffer, pH 7.4, room temperature. Sodium was extracted in double-distilled water and measured by atomic absorption spectrophotometry (Perkin-Elmer 1100B). Intracellular Na⁺ content was expressed as nanomoles of Na⁺ per milligram of protein.

Biochemical Assays

Renal Na,K-ATPase Activity. Rats were anesthetized with ether and then sacrificed by decapitation. Kidneys were removed, weighed, and sliced, and the outer medullas were dissected under stereomicroscope at 4°C, pooled, weighed, frozen in liquid nitrogen, and stored at 70°C up to the time of microsome preparation. Kidney outer medulla slices were suspended (1 g/10 ml) in an ice-cold solution containing 250 mM sucrose, 30 mM histidine, and 5 mM disodium EDTA (Sigma) at pH 7.2, and homogenized in a polytron (PCU-Kinematica AG, Lucerne, Switzerland) for 15 s at setting 5. The homogenate was centrifuged at 6000g for 15 min at 4°C (J2-21 M/E; Beckman Instruments), the supernatant fluid was decanted and saved, and the pellet was resuspended in the same solution, homogenized, and centrifuged at 6000g for 15 min at 4°C. The second supernatant was decanted, pooled with the first one, and centrifuged at 48,000g for 30 min at 4°C. Pellets were resuspended 1:1 (w/v) in the sucrose-histidine solution. The protein content of the microsomes was determined by Lowry's method (Lowry et al., 1951) using BSA as standard. Na,K-ATPase activity was determined in microsome preparations previously permeabilized with deoxycholic acid (0.65 mg of deoxycholic acid/mg protein, pH 7.4) for 30 min at room temperature. Na,K-ATPase activity was assayed in kidney microsomes after the release of inorganic ³²P from [³²P]ATP, as described previously (Ferrandi et al., 1996). Na,K-ATPase activity was calculated as ouabain-sensitive fraction of total ATPase activity and expressed as µmoles of P_i per minute per milligram of protein.

Plasma OLF Concentration. OLF was extracted from plasma and measured by radioimmunoassay, as already described (Ferrandi et al., 1997a). Plasma OLF concentrations were expressed in naomolar ouabain-equivalent according to a ouabain standard curve.

Renal Na,K-ATPase mRNA Levels. Renal 1 Na,K-ATPase mRNA levels were measured in pools of renal outer medullas from five rats for each treatment group according to the following procedures: RNA was prepared from rat renal medullas by the guanidine isothiocyanate-cesium chloride method (Chirgwin et al., 1979). Equal amounts of total RNA (10 µg) were size fractionated by electrophoresis on denaturing 1.3% agarose formaldehyde gels and subsequently transferred in 20× standard saline citrate (SSC) (1× SSC = 150 mM sodium chloride and 15 mM trisodium citrate) to GeneScreen nylon membrane (DuPont, Wilmington, DE). The total RNA amount was in the linear range of detection, i.e., between 5 and 30 µg. Hybridizations were performed for 16 h at 44°C in hybridization buffer (50% deionized formamide, 3× SSC, 10× Denhardt's solution, 0.1% SDS, and 100 mg/ml salmon sperm DNA), with excess of [³²P]2'-deoxycytidine 5'-triphosphate-labeled cDNAs encoding rat 1-Na,K-ATPase subunits and rat 18S probes using a multiprimer DNA labeling kit (Amersham, Arlington Heights, IL). The 1 cDNA probe consisted of a 2.2-kb NcoI restriction fragment. Membranes were washed in 2× SSC (5 min, room temperature) followed by increases in wash stringency up to 0.2× SSC-0.1% SDS (50 min at 65°C). Before rehybridization with other probes, cDNA probes were removed from membranes by washing with 1% SDS for 1 h at 70°C. Hybridization of RNA to a radiolabeled rat 18S cDNA probe served to confirm uniform amounts of total RNA in each lane. Na,K-ATPase 1 mRNA was visualized and quantified by electronic autoradiography (InstantImager 2024; Packard Instruments, Meriden, CT). RNA samples from different renal medulla pools were analyzed simultaneously on the same nylon filter. Quantities were expressed as arbitrary units normalized for the levels of 18S rRNA and then related to the control sample, which was given the arbitrary value of one.

Statistics

Data are reported as mean ± S.E.M. We performed factorial one-way ANOVA followed by Fisher's least squares difference test to evaluate the differences between different compound concentrations in cell culture studies. Factorial ANOVA for repeated measures was done to test the interaction of time and treatment or strain and treatment on the variables in each model. Factorial one-way ANOVA was then carried out to test the different groups versus the control group at different times and different times versus baseline time in each group. Dunnett's test was used to determine significance of the F ratio; p < .05 was considered significant for all comparisons.

	Results

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PST 2238 In Vivo Experiments

Long-Term Antihypertensive Activity Of PST 2238 in MHS and MNS Rats. The first study demonstrated that once-daily oral administration of PST 2238 (1, 3, and 10 mg/kg) to young prehypertensive MHS rats produced a reduction of the age-dependent SBP increase at all of the tested doses when recorded 6 h after a treatment (Fig. 1A). After 5 weeks of treatment, SBP in treated MHS rats was reduced compared with MHS controls. The reduction was similar for all of the tested doses, and no clear dose-dependence was observed. SBP recorded directly at the carotid at the end of the fifth week of treatment was similar to that recorded at the tail in both control and treated rats and was significantly reduced in MHS rats treated with either 1 or 10 mg/kg PST 2238 (Fig. 1A, inset). The antihypertensive response to PST 2238 was maintained even up to 24 h after treatment (Fig. 1B). PST 2238 had no effect on HR (Fig. 1C). Body weight (g) was not affected by PST 2238 treatment (fifth week, controls = 287.8 ± 9; 1 mg/kg = 281.4 ± 8; 3 mg/kg = 275.7 ± 6; 10 mg/kg = 283.6 ± 10.2).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Blood pressure lowering effect of PST 2238 in conscious MHS rats. The drug was administered orally, once a day, to young prehypertensive rats, starting from time 0 for 5 weeks. SBP was recorded weekly at the tail both 6 (A) and 24 h (B) after a treatment. SBP was recorded directly at the carotid in conscious rats 6 h after the last treatment (A, inset). HR was recorded weekly 6 h after a treatment (C). Data are mean ± S.E.M. of seven rats for each group. *p < .05; **p < .01 versus MHS control rats.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Time course and dose-dependent blood pressure lowering effect of PST 2238 in conscious MHS rats. Drug was administered orally, once a day, to young prehypertensive rats for 6 weeks. SBP was recorded weekly at the tail 6 h after a treatment. Data are mean ± S.E.M. of seven rats for each group. *p < .05; **p < .01 versus MHS control rats.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   SBP (A) and renal Na-K ATPase activity measured in outer medulla microsomes (B) of MHS and MNS rats after 6 weeks of oral treatment with vehicle (0.5% v/v methylcellulose, controls) or PST 2238 at 0.1, 3, and 90 µg/kg and 3 mg/kg (MHS) and PST 2238 at 90 µg/kg and 3 mg/kg (MNS). Data are mean ± S.E.M. of seven MHS and MNS rats for each group. *p < .05; **p < .01 versus MHS control rats. §p < .05; §§p < .01 versus MNS control rats.

Effect of PST 2238 on Renal Na,K-ATPase Activity and Plasma OLF Content. In the above second study, we verified whether the antihypertensive effect of PST 2238 was associated with modifications of renal Na,K-ATPase activity. Figure 3B shows that control MHS rats had significantly higher renal Na,K-ATPase activity than MNS controls, as already demonstrated (Ferrandi et al., 1996). PST 2238, at all of the tested doses, reduced renal Na,K-ATPase activity at V_max of MHS rats, with a significant effect at doses of 90 µg/kg and 3 mg/kg (Fig. 3B, left). At these high doses, renal Na,K-ATPase activity of MHS rats was completely normalized to the level of MNS controls. On the contrary, PST 2238 did not affect the renal Na,K-ATPase activity in MNS rats (Fig. 3B, right).

Effect of PST 2238 on Renal Na,K-ATPase Expression. It was previously demonstrated that the increased renal Na,K-ATPase activity of MHS rats is associated with higher mRNA levels of the enzyme than in MNS rats (Ferrandi et al., 1996). We therefore investigated whether the down-regulation of renal MHS Na,K-ATPase activity at V_max produced by long-term administration of PST 2238 was paralleled by a reduction of mRNA levels of the catalytic 1 Na,K-ATPase subunit. For technical reasons it was not possible to quantify 1 Na,K-ATPase mRNA levels from single rat kidneys; therefore, three pools of renal outer medullas, each obtained from five rats, were utilized for two treatment groups (controls and 90 µg/kg PST 2238 per os.). After 6 weeks of treatment, SBP was 164 ± 1.4 and 146.6 ± 1.26 mm Hg (p < .01) in MHS rats and 141.3 ± 1.24 and 139.3 ± 0.83 mm Hg in MNS rats receiving either vehicle or 90 µg/kg PST 2238, respectively. HR and body weight were not modified by the treatment in either strain (data not shown). Renal 1 Na,K-ATPase mRNA levels were significantly higher in adult MHS controls than in age-matched MNS controls (Fig. 4). Long-term PST 2238 treatment produced a statistically significant reduction of 1 mRNA levels in MHS rats (Fig. 4) but did not significantly affect mRNA levels in MNS (Fig. 4).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   1 renal Na,K-ATPase mRNA levels in adult MHS and MNS rats: effect of a 6-week oral treatment with 90 µg/kg PST 2238. Comparison of 1 mRNA levels between MHS and MNS control rats and effect of PST 2238 on 1 mRNA levels in MHS and MNS rats. Data are mean ± S.E.M. of three pools of five rats each, run in triplicate. *p < .05, significantly different from the respective control.

Long-Term Antihypertensive Activity of PST 2238 in MHS and SHR. A relatively high dose of PST 2238 (10 mg/kg) was chosen to compare its antihypertensive effect in two different animal models of genetic hypertension, MHS and SHR rats. K-canrenoate (60 mg/kg per os) was used as the reference compound. It has been previously demonstrated that MHS but not SHR rats respond to the latter antihypertensive treatment (Ferrari et al., 1993). PST 2238 and K-canrenoate caused a similar significant reduction in the development of hypertension when administered orally to young MHS prehypertensive rats for 5 weeks (Fig. 5A). However, neither compound had any effect on SBP of SHR rats (Fig. 5B).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Blood pressure lowering effect of PST 2238 and K-canrenoate in conscious MHS (A) and SHR rats (B). Drugs were orally administered to young prehypertensive rats starting from time 0 for 5 weeks. SBP was recorded weekly at the tail 6 h after a treatment. Data are mean ± S.E.M. of seven rats for each group. *p < .05; **p < .01 versus respective control rats.

Short-Term Antihypertensive Activity of PST 2238 in MHS Rats. The short-term effect of PST 2238 treatment on SBP of adult hypertensive MHS rats is reported in Fig. 6. Baseline SBP was 167 ± 2.5 mm Hg and was gradually lowered by PST 2238 at all of the tested doses. The maximal hypotensive effect was achieved with the highest dose (10 mg/kg) after 4 days of treatment. Then, starting from the 8th to the 10th day, a stable and similar reduction of SBP was observed at all doses. After treatment suspension, SBP gradually returned to the levels of MHS controls.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Blood pressure lowering effect of PST 2238 in adult conscious MHS rats. Drug was orally administered to adult (60 days of age) rats, starting from time 0 for 10 days. SBP was recorded daily, except during the weekend, 6 h after a treatment and during an additional 5-day period of washout. Data are mean ± S.E.M. of groups of 10 animals (5 in the 0.1-mg/kg PST 2238 group). *p < .05; **p < .01 versus controls.

Cell Culture Experiments

Effect of PST 2238 on the Na-K Pump of Cultured Rat Renal Cells. The transfected stable cell line NRK-1, overexpressing the hypertensive -adducin variant (F316Y), is characterized by an increase of Na-K pump surface expression (Tripodi et al., 1996) and activity at V_max (Tripodi et al., 1996) compared with the normal NRK-52E cell line (Fig. 7). To verify whether PST 2238 could modulate Na-K pump activity in cultured cells, both normal NRK-52E and transfected NRK-1 cells were incubated for a relatively long period of time (5 days) with very low concentrations (10¹²-10¹⁰-10⁹ M) of PST 2238. This experimental design was based on previous evidence that PST 2238 from 10¹² to 10⁸ M antagonizes the ouabain-dependent increase of the Na-K pump activity in normal NRK-52E cells after 5 days of incubation (Ferrari et al., 1998). Intracellular Na (nmol/mg), after load, was similar in both NRK-1 (347 ± 18.5) and NRK-52E control cells (321 ± 9) and was not affected by PST 2238 treatment (NRK-1: 10¹² M = 344 ± 32.3; 10¹⁰ = 305 ± 17; 10⁹= 331.9 ± 19; NRK-52E: 10¹² M = 306 ± 11; 10¹⁰ = 302.3 ± 16.5; 10⁹= 339.9 ± 17.4). As shown in Fig. 7, PST 2238 reduced Na-K pump activity at V_max of NRK-1 but not of normal NRK-52E cells, thus nullifying the difference in the maximal pump activity between these two cell lines.

View larger version (53K): [in this window] [in a new window]   Fig. 7.   Effect of 5-day incubation of both NRK-1 and NRK-52E wild-type rat renal cells with PST 2238 (1012-1010-109 M) on Na-K pump rate at Vmax. Data are mean ± S.E.M.; numbers are reported in the columns. *p < .05; **p < .01 versus NRK-1 control.

	Discussion

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PST 2238 is a new antihypertensive compound with an original mechanism of action: it antagonizes the increased blood pressure and renal Na,K-ATPase activity in experimental hypertension induced by chronic infusions of low doses of ouabain (Ferrari et al., 1998). In the present study, we have also demonstrated that, in a genetic form of rat hypertension sustained by renal Na-K pump hyperactivation and elevated OLF levels, PST 2238 lowered blood pressure and normalized the renal Na-K pump alteration.

Chronic treatment of young prehypertensive MHS rats with PST 2238 significantly reduced the age-dependent SBP increase even at doses of 1 and 10 µg/kg per os. This antihypertensive effect was not accompanied by variations in either HR or body weight and was characterized by a slow onset without signs of tachyphylaxis, as demonstrated by a stable 24-h SBP reduction observed even after 4 and 5 weeks of treatment. PST 2238 not only reduced the development of hypertension after long-term treatment, but also lowered SBP when given to already hypertensive adult MHS rats for a relatively short period of time (10 days). The hypotensive activity of PST 2238 in adult MHS rats took some days to reach the maximum effect and reverted 1 to 5 days after suspension of the treatment according to the dose administered. The antihypertensive activity of PST 2238 was also associated with a cellular effect as, in chronically treated MHS rats, it reduced the increased renal Na,K-ATPase activity as well as 1 mRNA levels, whereas it did not affect either blood pressure or Na,K-ATPase in MNS rats. This suggests that the increased expression of MHS renal Na-K pump and the consequent faster ion transport across renal epithelium are prevented or reduced by PST 2238.

The up-regulation of the renal Na-K pump in MHS rats may result from two distinct combined mechanisms: 1) a genetic abnormality of the cytoskeletal protein adducin (Bianchi et al., 1994) which by affecting the actin-cytoskeletal (Tripodi et al., 1996), induces increased Na-K pump surface expression and activity (Tripodi et al., 1996) and thus triggers faster transtubular ion transport (Ferrari et al., 1995), and 2) enhanced secretion of OLF (Ferrandi et al., 1992; Ferrandi et al., 1997a) which may sustain the up-regulation of the renal Na-K pump, as already demonstrated in ouabain-hypertensive rats (Ferrari et al., 1998). These findings raised the question of whether PST 2238 would normalize renal Na-K ATPase activity in vivo by direct modulation of the cellular mechanisms affected by the adducin alteration, by antagonizing OLF functional effects on Na,K-ATPase expression, or by a combination of both phenomena. In an attempt to answer this question and to exclude the possible involvement of circulating OLF (or ouabain), we assessed the effect of long-term exposure to PST 2238 on Na-K pump activity of both NRK cells (wild-type) and renal cells transfected with mutated adducin. We found that PST 2238 10¹⁰ and 10⁹ M counteracted the increased V_max Na-K pump activity caused by transfection with mutated adducin but did not modify the pump in wild-type cells, strongly suggesting a direct effect of PST 2238 on the altered cellular mechanisms regulating the Na-K pump expression in transfected cells. Moreover, we have previously observed that PST 2238 (from 10¹² M to 10⁸ M) prevents the increase of V_max Na-K pump activity induced in cultured NRK cells by ouabain and lowers in vivo blood pressure in rats and normalizes their renal Na,K-ATPase activity, both increased by long-term ouabain infusion (Ferrari et al., 1998). It seems therefore that both a direct modulation of the renal Na-K pump and antagonism of the OLF- (or ouabain) induced cellular effects could explain the antihypertensive activity of PST 2238 in both MHS and ouabain-dependent rat hypertension.

The lack of normalization of blood pressure in MHS rats treated with low doses of PST 2238 is not surprising considering that adducin polymorphism accounts for only 40 to 50% of the blood pressure difference between MHS and MNS rats (Bianchi et al., 1994). This is a common finding in other genetic animal models in which a single genetic mechanism accounts for only a portion of the blood pressure difference between the two parental strains.

Although a precise definition of the molecular mechanism by which PST 2238 modulates renal Na,K-ATPase in MHS rats is not yet available, the following working hypotheses may be considered:

PST 2238 may interfere directly with the interaction between adducin and Na,K-ATPase. We have recently demonstrated that in a cell-free system adducin interacts at nanomolar concentrations with purified Na,K-ATPase, favoring its conformational transition. This effect is produced by the hypertensive variant with higher affinity than that of normal adducin (Ferrandi et al., 1997b). We are currently evaluating what effect PST 2238 may have on this interaction.

PST 2238 may counteract the effect of mutated adducin on Na,K-ATPase at the transcriptional level. We have previously demonstrated that mutated adducin is associated both in vivo (Ferrandi et al., 1996) and in transfected rat renal cells (Tripodi et al., 1996) with an increase of V_max Na,K-ATPase activity. Moreover, the up-regulation of renal Na,K-ATPase activity in MHS is due to its increased expression (Ferrandi et al., 1996). As PST 2238 is able to down-regulate not only renal Na,K-ATPase activity, but also its mRNA level in MHS rats, it is suggested that this compound may normalize expression of the enzyme in hypertension by direct interference at the transcriptional level.

PST 2238 may affect the process of membrane insertion, internalization, and degradation of Na-K pumps, which determines the level of expression of this enzyme at the cell surface. It is known that both cell surface polarity (Hammerton et al., 1991; Mays et al., 1995) and long-term maintenance of the pump on the cell membrane are regulated by its interaction with the spectrin-actin cytoskeleton (Nelson and Veshnoc, 1987; Cantiello, 1995). Because adducin stabilizes the spectrin-actin interaction (Hughes and Bennett, 1995), it may play an important role in regulating the cycle of membrane skeleton assembly-disassembly and hence the half-life of Na,K-ATPase on the membrane. Studies are in progress to verify this hypothesis and to determine what influence PST 2238 may have on these processes.

The ability of PST 2238 to normalize Na,K-ATPase activity in MHS, ouabain-induced hypertensive rats, NRK cells transfected with hypertensive adducin, and NRK cells in which Na,K-ATPase activity is increased as a consequence of long-term ouabain incubation raises the question of whether both mutated adducin and ouabain (or OLF) affect Na,K-ATPase expression and activity (and consequently cause hypertension) through a common mechanism that is corrected by PST 2238. The number of Na-K pump units on cell membrane is determined by the rate of synthesis, insertion, and removal of these units. Because ouabain (and probably also adducin, our unpublished results) affects the half-life of cell membrane Na-K pumps (Rayson, 1989) by a combined mechanism of transient increase of the rate of synthesis (Tang and McDonough, 1992) and decrease of the pump degradation rate (Rayson, 1989), an increased transcription rate of the Na,K-ATPase may be proposed as the common mechanism triggered by adducin or ouabain that is affected by PST 2238.

The antihypertensive activity of PST 2238 seems to be selective for some forms of genetic hypertension, as we have demonstrated here that this compound has no effect on blood pressure of SHR rats. As already reported (Ferrari et al., 1993), also K-canrenoate shows a similar selectivity of action. These findings could be explained by the different behavior of renal Na,K-ATPase in the two strains. In fact, unlike MHS rats, SHR rats show a lower expression of Na,K-ATPase 1 subunit in the kidney than WKY-normotensive controls, both before and after the development of hypertension (Doris et al., 1997). Moreover, circulating OLF levels seem little involved in SHR hypertension, because they have been found reduced in SHR as compared with WKY (Doris, 1994, Doris et al., 1997). Finally, because both normotensive WKY and hypertensive SHR rats carry the same genetic variant of adducin as MHS rats (Tripodi et al., 1997) and because genetic hypertension develops as a polygenic disease where specific epistatic interactions lead to the final blood pressure rise, it is likely that the genetic background responsible for SHR hypertension is different from that operating in MHS. As PST 2238 seems to lower blood pressure specifically through a modulation of the renal Na,K-ATPase alteration, this may explain the lack of response of SHR rats.

In human -adducin, gene mutations are associated with hypertension (Casari et al., 1995; Cusi et al., 1997) and with a greater change in blood pressure after variation of body sodium (Cusi et al., 1997). It may be hypothesized that PST 2238 exerts a greater antihypertensive effect in patients carrying an -adducin gene mutation.