This study investigated the importance of the male sex hormone testosterone on salt-induced hypertension, renal α2-adrenoceptor subtype distribution, and gene expression in salt-sensitive (SBH) male Sabra rats. Comparisons of blood pressure and renal α2-adrenoceptor subtype gene expression and receptor densities have been made among sham-operated rats, and gonadectomized rats treated or not with testosterone and submitted to normal or high salt diet for 6 weeks. In intact rats, only α2B-adrenoceptors were detected in this rat strain independent of the diet. In these rats, high salt diet increases blood pressure and up-regulates gene expression and density of α2-adrenoceptors. Gonadectomy abolishes the hypertensive response to salt overload, decreases gene expression and density of α2B-adrenoceptors, and prevents their salt-induced up-regulation. After gonadectomy, increased gene expression and a detectable density of α2A-adrenoceptors are observed at similar levels in normal and high salt diet. In gonadectomized rats, testosterone replacement restores salt-induced hypertension, density of renal α2B-adrenoceptors, and gene expression to the intact levels observed both under normal and high salt diet. Furthermore, the α2A-adrenoceptor subtype is not detected in these conditions. If the increase in renal α2B-adrenoceptor subtypes is indicative of the hypertensive phenotype, the presence of the α2A-adrenoceptor appears associated with a state of salt resistance in male SBH rats. In conclusion, testosterone is needed for the full expression of salt-induced hypertension in male salt-sensitive Sabra rats. Renal densities of α2-adrenoceptor subtypes are under control of the testicles and are differentially regulated by testosterone.
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 α2-adrenoceptors in the modulation of water clearance and sodium excretion (Strandhoy, 1985; Gellai and Ruffolo, 1987). Renal α2-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 α2-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 α2-adrenoceptors in the development of hypertension have considered the possible role of sex. More recently, however, it has been demonstrated that both renal α2-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 α2-adrenoceptor density in SHR rats. The α2-adrenoceptor family includes different subtypes classified according to their selectivity toward different ligands (Bylund, 1992). In the kidney of the normotensive rat, α2A- and α2B-adrenoceptor subtypes have been characterized by binding studies (Uhlen and Wikberg, 1991). In SHR rats, testosterone regulates renal α2B-adrenoceptor density at the mRNA level (Gong et al., 1995). From this study, it has been postulated that the effects of testosterone on renal α2B-adrenoceptor mRNA and α2B-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 α2A-adrenoceptors. Recently, however, SHR rats were reported to have a defective ability of the α2A-adrenoceptor to increase renal solute excretion (Intengan and Smyth, 1997b). From this observation, it was thus suggested that an altered renal α2A-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 α2A- and α2B-adrenoceptors are detectable in renal membranes. However, in the male SBH rats, only the α2B-adrenoceptor is detected (Le Jossec et al., 1995). The latter observation has led to the suggestion that in SBH rats the presence of the α2B-adrenoceptor and/or the lack of α2A-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 α2-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 α2-adrenoceptor subtypes in the male salt-sensitive Sabra rat.
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.
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 α2-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 α2A-adrenoceptor agonist, or 0.1 nM to 1 mM prazosin, which is selective for α2B-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 α2-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 α2-adrenoceptor subtype cDNA. Primers used for amplification were as follows: α2A-, 5′ primer: 5′-GCGCCCCAGAACCTCTTCCT-3′ and 3′ primer: 5′-AGTGGCGGGAAGGAGATGAC-3′ (Chalberg et al., 1990); and α2B-, 5′ primer: 5′-AGCATCGGATCTTTTTTTGC-3′ and 3′ primer: 5′-GTTTGGGGTTCACATTCTTC-3′ (Le Jossec et al., 1995). For β-actin, similar experimental conditions were used, except that the annealing temperature was 53°C. Primers used for β-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 α2A-, α2B-, and β-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 α2A- and α2B-mRNA levels were expressed versus β-actin mRNA content.
[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).
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 χ2 and Kolmogorov-Smirnov tests.P < 0.05 was considered statistically significant.
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 α2-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 α2-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 α2-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 (Table2).
The inhibition of [3H]RX821002 binding by subtype-selective drugs was studied to determine whether the kidney of the different experimental groups showed differences in the α2-adrenoceptor subtype distribution. In renal membranes of both intact rats under normal and high salt diet, guanfacine (selective for α2A-adrenoceptor) (Fig. 2, top) and prazosin (selective for α2B-adrenoceptor) (Fig. 2, bottom) inhibited [3H]RX821002 binding with steep monophasic competition curves. In all of the experiments the curves were fit best to a model of interaction with a single class of α2-adrenoceptors having higher affinity for prazosin than for guanfacine (Table 2). These data were in agreement with the general characteristics of the α2B-adrenoceptor subtype. As a consequence, the α2A-adrenoceptor subtype was undetectable in intact SBH rats and the increase in α2-adrenoceptor density observed under high salt diet in these rats resulted only from a rise in the α2B-adrenoceptor subtype. In contrast, in gonadectomized rats the inhibition curves for guanfacine and prazosin (Fig. 2) were shallow, suggesting interaction of these compounds with a heterogeneous population of α2-adrenoceptors regardless of the diet. Analysis of these competition curves with the iterative curve-fitting program revealed in these renal membranes a major component of binding sites exhibiting (Table 2) high affinity for prazosin and a minor component having high affinity for guanfacine and corresponding to α2B- and α2A-adrenoceptor subtypes, respectively. From these results, the calculated α2A- and α2B-adrenoceptor densities in gonadectomized SBH under normal salt diet were 43.1 ± 16.8 and 146.6 ± 9.5 fmol/mg of protein, respectively. Interestingly, these densities in gonadectomized rats were unchanged by high salt diet. Therefore, these results revealed a marked decrease in α2B-adrenoceptor density (P < 0.001) compared with intact rats regardless of the regimen. After testosterone treatment of gonadectomized SBH rats under normal or high salt diet, inhibition curves for guanfacine and prazosin were steep and monophasic (Fig. 2), suggesting only one component of binding having characteristics of the α2B-adrenoceptor subtype (Table 2). As observed in intact animals, the increase in α2-adrenoceptor density induced by high salt diet in these rats resulted only from a raise in the α2B-adrenoceptor subtype.
Renal α2-Adrenoceptor Subtype Gene Expression.
Experiments using reverse transcription-polymerase chain reaction (RT-PCR) were performed with specific α2-adrenoceptor subtype primers to determine whether alterations in renal α2-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 β-actin, α2B-, and α2A-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 α2A-adrenoceptor (Fig.4, top) and 407 bp for the α2B-adrenoceptor (Fig. 4, middle)] in the kidney of all SBH rats regardless of the diet. On the other hand, the fragment generated from β-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 α2-adrenoceptor products normalized to β-actin revealed (Fig. 5) increased α2B- (P < 0.01) but equivalent α2A-mRNA levels in intact SBH rats compared with high salt and normal diet. Compared with intact rats, a strong increase (P < 0.001) in α2A-mRNA levels was observed in gonadectomized animals, whereas those for α2B-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.
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 α2A- and α2B-adrenoceptors were detectable in renal cortical membranes. However, only the α2B-adrenoceptor was detected in the male SBH Sabra rat (Le Jossec et al., 1995). We hypothesized that this altered distribution in renal α2-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 α2B-adrenoceptor subtype was found in the whole kidney of intact SBH rats. In addition, α2B-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 α2A-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 α2A-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 α2B-adrenoceptor mRNA, α2A-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 α2A-adrenoceptor, overexpression of the encoding gene, and abolished salt-induced high blood pressure. In contrast, renal α2B-adrenoceptor densities and mRNA were decreased to levels lower than those in intact rats under normal salt diet. Moreover, α2A- and α2B-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 α2B-adrenoceptors are found in kidney regardless of the diet. Interestingly, testosterone replacement restores high renal α2B-adrenoceptor density, the salt-induced increase of these receptors, and decreased α2A-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 α2-adrenoceptors gene expression and subtype distribution are androgen dependent in male SBH rats.
A role for the α2B-adrenoceptor in the development of high blood pressure is now strengthened by the following recent studies. In α2B-adrenoceptor knockout mice, a lack of immediate hypertensive response to α2-adrenoceptor agonists has been observed (Link et al., 1996). Moreover, mice lacking a full complement of the α2B-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 α2B-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 α2A-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 α2A-adrenoceptors in the kidney of SBH rats and prevents the salt-induced high blood pressure. Interestingly, we have previously reported the presence of α2A-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 α2A-adrenoceptor subtype by the selective α2A-agonist guanfacine was recently shown to increase urine flow rate solely by increasing osmolar clearance (Intengan and Smyth, 1997a). Conversely, the ability of the α2A-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 α2A-adrenoceptors. Based on the natriuretic activity of the renal α2A-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, α2A-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 α2-adrenoceptor subtypes is necessary to give definitive validation of this hypothesis in Sabra rats.
In conclusion, our results show that both renal α2-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 α2B-adrenoceptor density in this rat strain. Conversely, the presence of renal α2A-adrenoceptors in gonadectomized SBH rats underlines the potential role of this receptor subtype in the resistance to salt-induced hypertension in Sabra rats.
This study was supported by grants from INSERM and the University René Descartes.
- spontaneously hypertensive rats
- Sabra salt-sensitive
- Sabra salt-resistant
- polymerase chain reaction
- base pair
- reverse transcription-polymerase chain reaction
- Received July 12, 2001.
- Accepted September 19, 2001.
- The American Society for Pharmacology and Experimental Therapeutics