Introduction

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The kidney plays a major role in the chronic regulation of blood pressure via modulation of sodium and water excretion (Hall et al., 1990; Guyton, 1991). Several studies have focused on the role of the renal ₂-adrenoceptors in the modulation of water clearance and sodium excretion (Strandhoy, 1985; Gellai and Ruffolo, 1987). Renal ₂-adrenoceptor densities are increased in spontaneously hypertensive rats of the Kyoto (Pettinger et al., 1982) and Milan (Parini et al., 1987) strains, and salt-sensitive Dahl (Pettinger et al., 1982) and Sabra rats (Parini et al., 1983) compared with their respective control strains. The density of renal ₂-adrenoceptors is also increased by dietary salt intake in SHR (Sanchez and Pettinger, 1981) and in salt-sensitive Dahl (Pettinger et al., 1982) and Sabra rats (Diop et al., 1984).

It is well known that blood pressure is higher in male than in female rats (Wexler and Greenberg, 1978) and humans (Wiinberg et al., 1995). This sexual difference in blood pressure is present in genetically hypertensive rats, including SHR (Ely and Turner, 1990; Chen and Meng, 1991; Ely et al., 1991; Chen et al., 1992), and the Dahl salt-sensitive (Dahl et al., 1975) as well as their normotensive control strains Wistar Kyoto (Ely et al., 1991) and the Dahl salt-resistant (Rapp and Dene, 1985), respectively. It has been shown that gonadectomy retards the development of hypertension in SHR rats (Iams and Wexler, 1977; Masubuchi et al., 1982; Chen and Meng, 1991). Surprisingly, none of the studies mentioned above dealing with the importance of renal ₂-adrenoceptors in the development of hypertension have considered the possible role of sex. More recently, however, it has been demonstrated that both renal ₂-adrenoceptor density and blood pressure are influenced by sex in SHR and Wistar Kyoto rats (Gong et al., 1994). In this latter study, it was shown that androgens are needed for the full expression of hypertension as well as for the increase of renal ₂-adrenoceptor density in SHR rats. The ₂-adrenoceptor family includes different subtypes classified according to their selectivity toward different ligands (Bylund, 1992). In the kidney of the normotensive rat, _2A- and _2B-adrenoceptor subtypes have been characterized by binding studies (Uhlen and Wikberg, 1991). In SHR rats, testosterone regulates renal _2B-adrenoceptor density at the mRNA level (Gong et al., 1995). From this study, it has been postulated that the effects of testosterone on renal _2B-adrenoceptor mRNA and _2B-adrenoceptor density are tightly associated with blood pressure and probably responsible for the hypertension in SHR rats. Nevertheless, this study has not considered the presence and the possible role of renal _2A-adrenoceptors. Recently, however, SHR rats were reported to have a defective ability of the _2A-adrenoceptor to increase renal solute excretion (Intengan and Smyth, 1997b). From this observation, it was thus suggested that an altered renal _2A-adrenoceptor function may play a causal role in the pathogenesis of hypertension in this rat strain.

The Sabra rat has been used as a model to study salt-induced hypertension (Ben-Ishay and Yagil, 1994). Sabra salt-sensitive (SBH) rats fed a regular diet, in contrast to Sabra salt-resistant (SBN) rats, have borderline hypertension at an early life stage, a slightly elevated blood pressure in adult life, and become invariably hypertensive in response to relevant stimuli such as a deoxycorticosterone acetate salt or a high sodium diet (Ben-Ishay and Yagil, 1994). On a normal diet, radioligand binding studies have shown that in male SBN rats, both _2A- and _2B-adrenoceptors are detectable in renal membranes. However, in the male SBH rats, only the _2B-adrenoceptor is detected (Le Jossec et al., 1995). The latter observation has led to the suggestion that in SBH rats the presence of the _2B-adrenoceptor and/or the lack of _2A-adrenoceptors in the kidney could contribute to the sensitivity to sodium, thus predisposing these animals to hypertension. Unfortunately, none of the studies mentioned above (Parini et al., 1983; Diop et al., 1984; Le Jossec et al., 1995) have considered the influence of androgens in this genetic form of rodent salt-induced hypertension. Because both renal ₂-adrenoceptor density and blood pressure are regulated by androgens in SHR rats (Gong et al., 1994), the aim of the present study was to determine whether gonads and testosterone in particular could have any influence on salt-induced high blood pressure, gene expression, and distribution of renal ₂-adrenoceptor subtypes in the male salt-sensitive Sabra rat.

	Experimental Procedures

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Animals. We used original SBH male Sabra rats bred at the Center de Sélection et d'Elevage des Animaux de Laboratoires (Orléans, France). Twenty-eight rats were gonadectomized at 3 weeks of age under pentobarbital (40 mg/kg) anesthesia. After gonadectomy, rats were divided in two groups and injected daily i.p. with either 10 mg/kg testosterone propionate in olive oil or vehicle alone. In parallel, 14 SBH rats of the same age were sham operated and injected daily with olive oil. At 4 weeks of age, rats were divided into two subgroups, housed in individual cages, and allowed free access to either normal (0.2%) or high (8%) NaCl laboratory chow. Water was given ad libitum and rats were maintained at a constant room temperature (24°C) on a 12-h light/dark cycle. After 6 weeks, systolic blood pressure was measured between 9 and 11 AM by using the tail-cuff method, with an electrosphygmomanometer (Physiograph MK-III; Narco-Bio-Systems Inc., Houston, TX) on unanesthetized, restrained rats warmed to 38°C for 10 min. At least five replicate blood pressure measurements were obtained on two consecutive days (10 measurements over 2 days). The average of all measurements for each rat was taken as representative of systolic blood pressure. Two days later, rats were killed by decapitation. Trunk blood was collected in heparinized tubes, centrifuged, plasma separated, and stored at 80°C for hormone determinations. Kidneys were carefully removed and rapidly frozen in liquid nitrogen. All experimental protocols were approved by the University Animal Use and Care Committee.

Hormonal Determinations. Plasma testosterone levels were measured by radioimmunoassay with a commercial kit provided by Amersham Pharmacia Biotech (Les Ullis, France) according to the protocol of the manufacturer.

Radioligand Binding Studies. Renal membranes were prepared from whole kidney and radioligand binding studies were performed, as previously described (Le Jossec et al., 1995), with the specific radiolabeled ₂-adrenoceptor antagonist [³H]RX821002. Briefly, 300 µg of renal membranes was incubated with 0.1 to 8 nM [³H]RX821002, 1 mM EDTA-K, 100 µM 5'-guanylylimidodiphosphate, 140 mM NaCl, and 50 mM Tris-HCl, pH 7.4, in a final volume of 300 µl for 45 min at 25°C. Reactions were stopped by dilution with ice-cold incubation buffer and rapid vacuum filtration through Whatman GF/C filters (Whatman, Maidstone, UK). The filters were washed twice with ice-cold incubation buffer and the radioactivity retained on filters was quantified by liquid scintillation counting. Nonspecific binding was determined in the presence of 20 µM phentolamine and represented 10% of the total binding. For competition studies, membranes were incubated for 45 min at 25°C with 2 nM [³H]RX821002 (a concentration near the K_d value) and either 0.1 nM to 1 mM guanfacine, a selective _2A-adrenoceptor agonist, or 0.1 nM to 1 mM prazosin, which is selective for _2B-adrenoceptors. The resultant saturation and competition curves were analyzed using a nonlinear least-squares curve-fitting program (Prism; GraphPad Software, San Diego, CA). Protein concentrations were determined according to Bradford (1976) by using bovine serum albumin as the standard.

Analysis of Renal ₂-Adrenoceptor mRNA. Total renal RNA was isolated using guanidium thiocyanate-phenol-chloroform extraction, with the TRIzol reagent (Invitrogen, Cergy-Pontoise, France) and used for RNA-directed complementary cDNA synthesis and DNA amplification as previously described (Le Jossec et al., 1995), except that HotStarTaq DNA polymerase (QIAGEN S.A., Courtaboeuf, France) was used according to the conditions provided by the supplier, and 1 µCi [³H]dCTP was added to the PCR reaction. PCR mixtures of cDNA and respective primers were amplified using a program temperature control system (Appligene Oncor, Illkirch, France). One cycle of PCR consisted of 1 min at 94°C, 1 min at 57°C, and 1 min at 72°C for ₂-adrenoceptor subtype cDNA. Primers used for amplification were as follows: _2A-, 5' primer: 5'-GCGCCCCAGAACCTCTTCCT-3' and 3' primer: 5'-AGTGGCGGGAAGGAGATGAC-3' (Chalberg et al., 1990); and _2B-, 5' primer: 5'-AGCATCGGATCTTTTTTTGC-3' and 3' primer: 5'-GTTTGGGGTTCACATTCTTC-3' (Le Jossec et al., 1995). For -actin, similar experimental conditions were used, except that the annealing temperature was 53°C. Primers used for -actin amplification (5' primer: 5'-TACAACCTCCTTGCAGCTCC-3'; 3' primer: 5'-ACAATGCCGTGTTCAATGG-3'; Nudel et al., 1983) were chosen to span two introns to discriminate the cDNA amplification products from genomic DNA contamination. Each reaction mixture was separated on a 1.5% low melting point agarose (Invitrogen) gel stained with ethidium bromide and documented on Polaroid 665 film (Polaroid UK Ltd., St. Albans, UK). For quantification, respective bands for _2A-, _2B-, and -actin signals were excised, agarose melted at 70°C, and the incorporated radioactivity determined by scintillation counting in Aquasafe 300 Plus (Zinsser Analytic, Frankfurt, Germany). The incorporated radioactivity was normalized with respect to the length of the three cDNA and _2A- and _2B-mRNA levels were expressed versus -actin mRNA content.

Materials. [³H]RX821002 (2.29 × 10¹² Bq/mmol), [³H]dCTP (1.92 × 10¹² Bq/mmol), and DNA molecular weight markers (100-bp ladder) were purchased from Amersham Pharmacia Biotech. Oligonucleotides were synthesized by Eurogentec (Herstal, Belgium). The following drugs were supplied by the indicated companies: prazosin (Pfizer Central Research, Sandwich, UK) and guanfacine (Novartis, Basel, Switzerland). All other materials were obtained from Sigma-Aldrich (Saint-Quentin-Fallavier, France).

Statistical Analysis. All results were expressed as the mean ± S.E.M. Statistical analyses were performed using analysis of variance followed by the Student-Newman-Keuls multiple comparison test. Data from DNA amplification were analyzed using the nonparametric one-way procedure of the SAS system (SAS Institute, Cary, NC), and comparison between groups was made using the ² and Kolmogorov-Smirnov tests. P < 0.05 was considered statistically significant.

	Results

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Physiological Data. After 6 weeks of a treatment regimen, blood pressures (Table 1) in intact rats were significantly increased by high sodium diet compared with normal diet. Gonadectomized rats exhibited blood pressures similar to those observed in intact controls under normal salt diet regardless of regimen. Testosterone replacement induced a slight increase of blood pressure in castrated rats under normal salt diet and restored high levels in sodium overload similar to those observed in intact animals. No significant differences in body weights were observed between the six groups of rats (Table 1). In contrast, kidney weights were decreased in castrated rats and reached similar control levels by testosterone replacement. Plasma testosterone levels were markedly decreased by gonadectomy, and testosterone treatment raised these levels to similar values as observed in intact rats. No differences in testosterone levels were found between normal and high salt diet.

Renal ₂-Adrenoceptor Densities and Subtype Distribution. Specific binding of [³H]RX821002 to renal membranes of all SBH rats was a saturable process (data not shown). Scatchard plots of [³H]RX821002 binding were monophasic, suggesting only one binding component (data not shown). In all experiments, Scatchard plots were best analyzed by a model with only one high-affinity class of sites regardless of diet. In Fig. 1, it was shown that in normal salt diet, gonadectomy and testosterone replacement did not change renal ₂-adrenoceptor binding capacities compared with intact rats. High sodium diet induced a marked increase in these binding capacities in intact rats. This dietary sodium-induced change in receptor density was not observed after gonadectomy. In contrast, this sodium-induced increase in renal ₂-adrenoceptor binding capacities was restored by testosterone treatment of gonadectomized rats. No differences in binding affinities were observed between intact, gonadectomized, and testosterone-treated rats and between normal and high salt diet (Table 2).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Renal [3H]RX821002 binding capacities of intact (Sham), gonadectomized (God), and gonadectomized treated with testosterone (God+T) SBH rats under normal () and high salt diet (). Results are expressed as mean ± S.E.M. (n = 7) of binding capacities (Bmax in fmol/mg of protein) determined as described under Experimental Procedures. **P < 0.01, ***P < 0.001 for comparison between normal and high salt diet.

View this table: [in this window] [in a new window]   TABLE 2 Binding characteristics of renal 2-adrenoceptors

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Guanfacine (top) and prazosin (bottom) compete for 2-adrenoceptor [3H]RX821002 binding sites on renal membranes of SBH rats under normal (left) and high salt diet (right). Data are representative of seven separate experiments and each experiment was conducted in duplicate. Total specific [3H]RX821002 binding capacities (Bmax in fmol/mg of protein) are extrapolated according to the relation Bmax= Bound × (Kd25°C + L)/L, where Bound is the capacity observed in the experiment in femtomoles per milligram of protein, Kd25°C is the equilibrium dissociation constant for [3H]RX821002 obtained from saturation experiments (in nM), and L the concentration of radioligand in the experiment (in nM).

Renal ₂-Adrenoceptor Subtype Gene Expression. Experiments using reverse transcription-polymerase chain reaction (RT-PCR) were performed with specific ₂-adrenoceptor subtype primers to determine whether alterations in renal ₂-adrenoceptor subtype distribution are associated with modifications in mRNA levels. In preliminary experiments, linearity of cDNA amplifications with these specific primers has been investigated on intact rats under normal and high salt diet (Fig. 3). From these results, totals of 28, 33, and 36 cycles of PCR are within the linear range of PCR for -actin, ₂B-, and ₂A-cDNAs, respectively, and consequently used in all experiments. In addition, cDNA amplifications with these specific primers show amplified products of the predicted size [311 bp for the _2A-adrenoceptor (Fig. 4, top) and 407 bp for the _2B-adrenoceptor (Fig. 4, middle)] in the kidney of all SBH rats regardless of the diet. On the other hand, the fragment generated from -actin primers (280 bp; Fig. 4, bottom), which is present at comparable levels in the six groups, is the only fragment amplified, ruling out any genomic DNA contamination. Analysis of the radioactivity incorporated in the ₂-adrenoceptor products normalized to -actin revealed (Fig. 5) increased _2B- (P < 0.01) but equivalent _2A-mRNA levels in intact SBH rats compared with high salt and normal diet. Compared with intact rats, a strong increase (P < 0.001) in _2A-mRNA levels was observed in gonadectomized animals, whereas those for _2B-mRNA levels decreased (P < 0.05). Therefore, similar mRNA levels were found between normal and high salt diet (Fig. 5). Testosterone replacement of gonadectomized rats restored a pattern similar to that observed in intact animals both in normal and high salt overload.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Linearity of cDNA amplifications using 2-adrenoceptor subtypes or -actin primers in kidney of intact SBH rats under normal () and high salt diet (). Results are representative of one experiments performed between 20 and 45 cycles of amplification.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Ethidium bromide staining of RT-PCR products using 2-adrenoceptor subtypes or -actin primers in kidney of SBH rats under normal and high salt diet. Results are representative of one experiment performed at 33 cycles of amplifications for 2-primers and 28 cycles for -actin. DNA size markers: lanes 1, intact SBH under normal (lane 2) and high salt diet (line 3); gonadectomized SBH under normal (line 4) and high salt diet (line 5); gonadectomized SBH treated with testosterone under normal (line 6) and high salt diet (line 7). Top, 2A-products. Middle, 2B-products. Bottom, -actin products.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Gene expression of 2-adrenoceptor subtypes determined by RT-PCR normalized to corresponding -actin levels and expressed as percentage of SBH values under normal diet. Data for normal () and high salt diet () are given as the mean ± S.E.M. (n = 7). *P < 0.05, **P < 0.01 for the comparison between normal and high salt diet; °P < 0.05, °°°P < 0.001 for the comparison between sham and gonadectomized; and +P < 0.05, ++P < 0.01, +++P < 0.001 for the comparison between gonadectomized and gonadectomized treated with testosterone.

	Discussion

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The results presented here show that gonadectomy prevented the hypertensive response to high salt diet in male SBH Sabra rats. Under these conditions, blood pressure was found to be similar to that observed in intact rats submitted to normal diet. In addition, the salt-induced high blood pressure was restored by chronic testosterone treatment of gonadectomized rats. From these results, it appears clear that testosterone is needed for the full expression of salt-induced hypertension in male SBH rats. It should be noted that our finding is in conflict with the recent study of Yagil and Yagil (2000) refuting the hypothesis that gonadal hormones modulate the blood pressure response to salt loading in the newly selected Sabra rat strain. Although these studies found different quantitative trait loci segregating with the blood pressure response to salt and suggested that gonads or sex hormones may contribute to salt sensitivity in SBHy (Yagil et al., 1998, 1999; Yagil and Yagil, 1998a), they failed to find any modification in salt-induced hypertension after gonadectomy (Yagil and Yagil, 2000) regardless of sex. The reason for this discrepancy could be the fact that we have used the original SBH rat strain, which may display some differences compared with the newly selected SBHy. An alternative explanation is that we have used 8% high salt diet as a mode of salt loading, whereas SBHy rats were treated with deoxycorticosterone acetate-salt (Yagil and Yagil, 2000). This treatment accelerates the onset of hypertension in SBHy rats (Yagil and Yagil, 1998b), and it is conceivable that the administration of exogenous mineralocorticoid in the form of deoxycorticosterone acetate leads to hypertensive response unrelated, in part, to high salt loading and consequently masking the testosterone dependence of salt-induced hypertension of this rat strain.

Previously, we have shown that in the male normotensive salt-resistant SBN rat, both _2A- and _2B-adrenoceptors were detectable in renal cortical membranes. However, only the _2B-adrenoceptor was detected in the male SBH Sabra rat (Le Jossec et al., 1995). We hypothesized that this altered distribution in renal ₂-adrenoceptor subtypes probably represented a genetic abnormality predisposing SBH rats to develop high blood pressure when submitted to high salt diet. In the present study, both under normal or high salt diet, only the _2B-adrenoceptor subtype was found in the whole kidney of intact SBH rats. In addition, _2B-adrenoceptor densities were markedly increased by the high salt diet. These high levels, which appear to be the consequence of an overexpression of the encoding gene, have also been observed in SHR rats (Gong et al., 1994, 1995). However, the _2A-adrenoceptor mRNA was clearly present in kidneys of intact SBH rats under both normal and high salt diet. These discrepant findings could be explained by the limited sensitivity of the binding technique, which is unable to detect low levels of _2A-adrenoceptor sites, if any, in SBH rats. Another explanation could be that posttranscriptional or posttranslational events occur in the kidney of SBH rats, preventing detection of the receptor protein by binding studies. In contrast to the ₂B-adrenoceptor mRNA, _2A-adrenoceptor mRNA levels were completely insensitive to the high salt diet in intact SBH rats. Surprisingly, the major findings of the present study were that gonadectomy induced pharmacological detection of the _2A-adrenoceptor, overexpression of the encoding gene, and abolished salt-induced high blood pressure. In contrast, renal _2B-adrenoceptor densities and mRNA were decreased to levels lower than those in intact rats under normal salt diet. Moreover, _2A- and _2B-adrenoceptor densities and expression of encoding genes were insensitive to the high salt diet in gonadectomized SBH rats. In gonadectomized rats treated with testosterone and as observed in intact animals, only _2B-adrenoceptors are found in kidney regardless of the diet. Interestingly, testosterone replacement restores high renal _2B-adrenoceptor density, the salt-induced increase of these receptors, and decreased _2A-adrenoceptor mRNA levels that correspond to the pattern found in intact SBH rats under both normal and high salt diet. These results provide clear evidence that renal ₂-adrenoceptors gene expression and subtype distribution are androgen dependent in male SBH rats.

A role for the _2B-adrenoceptor in the development of high blood pressure is now strengthened by the following recent studies. In _2B-adrenoceptor knockout mice, a lack of immediate hypertensive response to ₂-adrenoceptor agonists has been observed (Link et al., 1996). Moreover, mice lacking a full complement of the _2B-adrenoceptor gene are unable to raise blood pressure in response to chronic salt loading after subtotal nephrectomy (Makaritsis et al., 1999). However, from our study, it seems clear that renal _2B-adrenoceptors do not appear solely implicated in the hypertensive phenotype of male SBH rats. It is indeed possible that an association with a lack of adequately functional renal _2A-adrenoceptor may facilitate the onset of hypertension. Our present observations in gonadectomized SBH rats provide strong support for this hypothesis. In fact, gonadectomy induces the presence of pharmacologically detectable _2A-adrenoceptors in the kidney of SBH rats and prevents the salt-induced high blood pressure. Interestingly, we have previously reported the presence of _2A-adrenoceptors in renal cortical membranes of normotensive salt-resistant SBN rats (Le Jossec et al., 1995). It is thus conceivable to speculate on the importance of these receptors in the resistance to salt-induced hypertension in the Sabra rat strain. In normotensive rats, stimulation of the renal _2A-adrenoceptor subtype by the selective _2A-agonist guanfacine was recently shown to increase urine flow rate solely by increasing osmolar clearance (Intengan and Smyth, 1997a). Conversely, the ability of the _2A-adrenoceptor to mediate an increase in osmolar clearance was found lacking in SHR rats (Intengan and Smyth, 1997b). On a normal diet, urinary flow and sodium, potassium, and total solutes excretion are significantly lower in intact SBH compared with SBN rats (Ben-Ishay and Yagil, 1994). Together, these observations are consistent with the hypothesis that the impaired renal sodium excretion and salt sensitivity of SBH rats are related to the absence of renal _2A-adrenoceptors. Based on the natriuretic activity of the renal _2A-adrenoceptor shown in Wistar and Spague-Dawley rats (Intengan and Smyth, 1996, 1997a), it can reasonably be postulated that the lack of this receptor in the SBH rats may be contributing to the sensitivity to sodium and thus predisposing these animals to develop hypertension when submitted to high salt intake. In contrast, _2A-adrenoceptors are present and overexpressed in the kidney of gonadectomized SBH rats. This phenomenon could be a protective change against salt overload, resulting in an increased sodium excretion and the maintenance of normal blood pressure when submitted to high salt diet. However, functional study of the renal ₂-adrenoceptor subtypes is necessary to give definitive validation of this hypothesis in Sabra rats.

In conclusion, our results show that both renal ₂-adrenoceptor subtype distribution and salt-induced high blood pressure are influenced by androgens in male SBH rats. Testosterone is needed for the full expression of salt-induced hypertension and increased renal _2B-adrenoceptor density in this rat strain. Conversely, the presence of renal _2A-adrenoceptors in gonadectomized SBH rats underlines the potential role of this receptor subtype in the resistance to salt-induced hypertension in Sabra rats.