Testosterone reduces vascular relaxation by altering cyclic adenosine monophosphate pathway and potassium channel activation in male Sprague Dawley rats fed a high-salt diet

Abstract

Objectives:

Male gender and high-salt diet are risk factors for hypertension. The effect of chronic exposure to testosterone is an increase in vascular tone but its influence upon responses induced by other vasoactive agents is not clear. We considered the possibility of interactions between testosterone and a high-salt diet in the mechanisms that are involved in the regulation of vascular tone. Therefore, we designed experiments to assess the involvement of the cyclic adenosine monophosphate (cAMP) pathway and potassium channel activation on vascular relaxation elicited by testosterone deficiency that was induced by orchidectomy in Sprague Dawley rats on a normal or high-salt diet.

Method:

Weanling male rats were randomly divided into eight groups (n = 6 each) that were either orchidectomized or sham operated with or without testosterone replacement (10 mg/kg body weight of Sustanon 250 intramuscularly, Organon, Holland) and were placed on a normal or high-salt (0.3% or 8% NaCl) diet, respectively, for 6 weeks. Arterial blood pressure was determined before and weekly throughout the experiment using the tail-cuff method. Relaxation responses to forskolin and diazoxide were studied in noradrenaline (0.1 µM) precontracted aortic rings.

Results:

There was an increase in the systolic blood pressure of rats placed on a high-salt diet compared with control or orchidectomized rats. Orchidectomy elicited a reduction in the systolic blood pressure while testosterone replacement restored systolic blood pressure to values seen in intact rats. A high-salt diet reduced the relaxation response to forskolin and diazoxide but not in orchidectomized rats while testosterone replacement re-established the blunted relaxation response to forskolin and diazoxide.

Conclusion:

Inhibition of potassium channel or adenylyl cyclase activation appears to contribute to the mechanisms by which a high-salt diet increases vascular tone. These effects were counteracted by orchidectomy in male Sprague Dawley rats.

Keywords

adenylyl cyclase blood pressure diazoxide forskolin hypertension nucleotides orchidectomy vascular reactivity

Introduction

The role played by sex steroids in the regulation of vascular tone has been implicated as one of the contributing factors to gender dimorphism in cardiovascular diseases [Sader and Celermajer, 2000; Thompson and Khalil, 2003]. For example, noradrenaline elicits less forearm vasoconstriction in women than in men [Kneale et al. 2000] and contraction to noradrenaline and phenylephrine is greater in the aorta of intact male rats than intact female rats [Kneale et al. 2000], while oxidized low-density lipoprotein enhances 5-hydroxytryptamine-induced contraction to a greater extent in coronary arteries from male pigs than female pigs [Cox and Cohen, 1997]. Xia and Khalil reported a higher contractile response in the inferior vena cava of male rats than females rats and that Ca²⁺-dependent contraction and the myofilament contraction sensitivity to [Ca²⁺]_i are reduced in female rats compared with male rats [Xia and Khalil, 2010]. These gender differences have been primarily ascribed to the vascular protective effect of oestrogen [Liu et al. 2003], with fewer studies on the vascular effects of testosterone.

Hypertension is one of the most common cardiovascular diseases and a high-salt diet has been implicated in the pathogenesis of hypertension [Meneton et al. 2005]. However, salt-induced hypertension exhibits sexual differences because when fed a high-salt diet, male rats developed a higher blood pressure compared with female rats [Chappell et al. 2008]. One of the mechanisms by which a high-salt diet induces hypertension includes vascular function impairment. For instance, vascular dysfunction that is evident by impaired vasodilator response to dietary NaCl loading has been implicated as the initiating pressor effect in salt-sensitive individuals [Schmidlin et al. 2007]. Increased dietary salt intake has also been demonstrated to impair relaxation response to vasodilator agents in several vascular beds in laboratory animals [Nurkiewicz and Boegehold, 2007; Zhu et al. 2007]. Recently we reported the testosterone-dependent impairment effect of a high-salt diet on the endothelial function in a male rat [Oloyo et al. 2011].

Usually acetylcholine and sodium nitroprusside are the conventional pharmacological agents used in vasodilatory studies. However, Sofola and colleagues demonstrated an impaired arterial relaxation response to activation by forskolin of the cAMP pathway in rats fed a high-salt diet [Sofola et al. 2003]. Activation of adenylyl cyclase and consequently cAMP production causes vascular smooth muscle relaxation via several downstream mechanisms involving protein kinase A (PKA), intracellular calcium ion concentration modulation, potassium channel opening, among others [Taylor et al. 1999].

Ion channels are present on the plasma membrane and intracellular organelles of all cells and they play important roles, such as secretion of hormones and neurotransmitters and vascular smooth muscle contraction and relaxation in these cells. Potassium ion channels are the most abundant, having various important functions in the cardiovascular system such as the repolarization of the cardiac action potential and relaxation of smooth muscle [Sandhiya and Dkhar, 2009]. Potassium channels are grouped into families as a function of their physiology and pharmacology [Miller, 2000]. The adenosine triphosphate (ATP)-dependent potassium channel (K_ATP), a subgroup of the inward rectifier family, is one of the most studied potassium channels because of its importance in health and diseases [Polleselloa and Mehazaab, 2004]. K_ATP channels are present in the pancreas, heart, brain and smooth muscle. Diazoxide is a potassium channel opener [Newgreen et al. 1990]. Diazoxide attenuates blood pressure in people with hypertension and has been demonstrated to possess the potassium channel opening property in the rat vascular smooth muscle [Newgreen et al. 1990]. Pasdois and colleagues reported the cardioprotective effect of diazoxide as a mitochondrial-ATP sensitive potassium (mitoK_ATP) channel opener via prevention of reactive oxygen species generation in an ischaemic-reperfused rat [Pasdois et al. 2008]. Likewise, diazoxide has been shown to preserve hypercapnia-induced cerebral arteriolar dilation in rats [Domoki et al. 2005].

Previous studies have suggested that the effect of chronic exposure to testosterone is an increase in vascular tone. For instance, testosterone suppresses the endothelial-dependent dilation of the cerebral arteries in rat [Gonzales et al. 2004]. A previous study from our laboratory has also demonstrated the testosterone dependence of the vascular function impairment effect of a high-salt diet in rats [Oloyo et al. 2011]. However, the influence of testosterone responses induced by other vasoactive agents, especially in salt-induced hypertension, remains unclear. This study was therefore designed to assess the effect of testosterone on vascular relaxation responses to the cAMP pathway and K_ATP channel activation in male Sprague Dawley rats fed a high-salt diet.

Methods

The experimental protocol used for this study was approved by the Animal Research and Ethics Committee of the Biomedical Technology, Wing Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum Kerala, India.

Forty-eight weanling male Sprague Dawley rats were obtained from the Department of Laboratory Animal Sciences, BioMedical and Technology wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Kerala, India. The rats (8 weeks old) with weight range of 90–110 g were housed in steel cages and kept for 12 h in the light and 12 h in the dark. Food and water was provided ad libitum. The rats were divided into eight groups of six rats each. Groups I and II were intact rats, groups III and IV were orchidectomized rats, groups V and VI were rats given Sustanon (Organon, Holland) injection as testosterone replacement following orchidectomy, and groups VII and VIII were sham orchidectomized rats. For orchidectomy, rats were anesthetized with ketamine and xylazine (90 mg and 10 mg/kg/body weight intramuscularly) [Gonzales et al. 2004] respectively for bilateral removal of the testes under aseptic surgical conditions, while in groups VII and VIII, the scrotal sacs were opened and sutured back as a model of sham orchidectomy. All rats that had surgery received an injection of penicillin 300,000 iu/kg body weight at the time of surgery to prevent infections and were allowed a 3-day recovery period before the beginning of the experiments [Zhu et al. 2005]. After recovery from anaesthesia, all animals were returned to their cages. Rats in groups I, III, V and VII were fed with rat chow containing normal salt concentration (0.3% NaCl) while rats in groups II, IV, VI and VIII were fed with a high-salt diet (8% NaCl) and tap water ad libitum for 6 weeks. Group V and VI rats received 10 mg/kg body weight of testosterone suspension (Sustanon 250 intramuscularly) once in 3 weeks during the study for testosterone replacement.

Blood pressure monitoring

The conscious rats were placed in a restrainer on a heated pad (37°C) and allowed to adapt/rest inside for 15 min before blood pressure was measured weekly [Pojoga et al. 2008]. The rat tail was placed inside a 9 mm or 11 mm tail cuff, and the cuff was inflated and released several times to allow the animal to become conditioned to the procedure. Five consecutive blood pressure and heart rates measurements were obtained using the noninvasive blood pressure monitor MP35 (BIOPAC System Inc., Goleta, California, USA), which was connected to a computer. Blood pressure tracings were obtained through preinstalled software for BSL Pro.3.7.

Serum testosterone assay

Whole blood was collected via cardiac puncture using a 5 ml syringe and 21-gauge needle and serum was separated and stored at −80°C. Serum testosterone levels were measured by enzyme-linked immunoassay [Marcus and Durnford, 1986] using a commercial kit from Biotech Laboratories (Suffolk, UK) according to the protocol of the manufacturer. The kit uses the principle of competitive microplate enzyme immunoassay, whereby testosterone that is present in the sample competes with enzyme–testosterone conjugate for binding with a antitestosterone-coated microplate to form an antigen–antibody complex.

Isolation and preparation of aortic rings

At the end of the experimental period the rats were sacrificed by cervical dislocation. Thereafter the thoracic cage was opened and the aorta was cut at the visible ends and quickly placed in a Petri dish containing cold (4°C) Hepes buffered physiological salt solution (PSS). The aorta was carefully freed of connective tissue and the abdominal portion below the diaphragm was cut into 3 mm rings segment. The ring was then mounted between two fine stainless steel rods, with the small S-shape attached to a thread. The upper part of the rod was attached to the clamp of the micropositioner, while the thread was attached to the isometric force transducer (top force transducer MLT 050/D; AD Instruments, Bella Vista, New South Wales (NSW), Australia). The rings were superfused in 20 ml organ bath (Panlab LETICA series 01), with Hepes buffer solution at 37°C and gassed with 100% oxygen. The pH of the PSS was between 7.35 and 7.40, and all baths used simultaneously had a parallel connection to the source of PSS. The composition of the solution in mmol/liter was NaCl 133, KCl 3.6, CaCl₂ 1.8, MgCl₂.6H₂O 1.2, glucose 16, Hepes 3, and KH₂PO₄ 1.18.

Experimental protocol

After mounting the ring, a passive tension of 2 g was applied to each ring and then allowed to equilibrate for 90 min, during which period each ring was subjected to a submaximal dose (0.1 µM) of noradrenalin at 30 min intervals. Isometric tension was then measured using the top force transducer which was connected to a Powerlab 2/25 recorder (AD Instruments, Bella Vista, NSW, Australia).

To assess the role of the cAMP pathway on vascular reactivity, cumulative relaxation response curves of aortic rings to forskolin were obtained. Aortic rings were precontracted with 0.1 µM noradrenalin following which the rings were exposed to cumulative doses (0.01–10µM) of forskolin.

The possible involvement of the K_ATP channel in the effect of androgens on vascular reactivity was assessed by studying the aortic relaxation response to diazoxide. This was carried out across the groups. Aortic rings were precontracted with 0.1 µM noradrenalin, after which a cumulative dose (0.01–10 µM) of diazoxide was added to the organ bath.

Drugs and solutions and statistical analysis

Forskolin and diazoxide were dissolved and diluted in dimethylsulphuroxide. Unless otherwise mentioned, all chemicals and reagents were purchased from Sigma-Aldrich (Bangalore, India).

Relaxation responses to forskolin and diazoxide were expressed as percentage of the active tone achieved with 0.1 µM noradrenaline. The half maximal effective drug concentration (EC₅₀) was calculated for each concentration response curve. The pEC₅₀ was calculated as the negative logarithm to base 10 of the EC₅₀ using programmed statistical software (Graphpad Prism 5 USA). The collected data were expressed as mean ± standard error of the mean and were analyzed using one-way analysis of variance followed by the Student–Newman–Keuls test post hoc to identify differences between individual means.

Results

Blood pressure

Table 1 shows the systolic blood pressure (SBP) of the rats across the groups. After the 6-week experimental period, a high-salt diet significantly increased (p < 0.05) the SBP of the rats compared with those fed a normal-salt diet. The increase in SBP in the orchidectomy plus high-salt diet group was significantly less (p < 0.05) compared with the intact plus high-salt diet group. Testosterone replacement almost restored SBP to the level observed in intact animals as the increase in SBP of rats in the testosterone replacement groups was significantly higher (p < 0.05) compared with their corresponding orchidectomized groups without testosterone replacement. Orchidectomy also significantly reduced (p < 0.05) SBP elevation even in rats fed a normal diet. Sham orchidectomy had no significant effect (p > 0.05) on the SBP in groups of rats on the normal diet and high-salt diet.

Table 1.

Systolic blood pressure and serum concentration of testosterone in the rats in all of the groups.

Groups n= 6	Systolic blood pressure (mmHg)	Serum testosterone concentration (nmol/liter)
INT + NS	130 ± 3.0	13.26 ± 0.32
INT + HS	154 ± 4.0^*	17.22 ± 0.81
ORCH + NS	120 ± 3.0	0.84 ± 0.24^‡
ORCH + HS	132 ± 4.0^$	2.30 ± 0.15^‡
ORCH + TES + NS	132 ± 3.0	11.54 ± 1.3
ORCH + TES + HS	146 ± 3.0^*	14.66 ± 1.70
SHAM + NS	133 ± 4.0	11.90 ± 1.35
SHAM + HS	150 ± 5.0^*	16.24 ± 1.20

Data presented as mean ± standard error of the mean.

Significant increase (p < 0.05) compared with corresponding control; ^$significant decrease (p < 0.05) compared with intact and high-salt group; significant decrease (^‡p < 0.001) compared with intact, testosterone replacement and sham orchidectomized groups.

HS, high salt; INT, intact; NS, normal salt; ORCH, orchidectomy; SHAM, sham orchidectomy; TES, testosterone.

Testosterone assay

Table 1 also shows the serum level of testosterone across the groups of rats. Orchidectomy significantly reduced (p < 0.001) the level of testosterone in the serum compared with the intact, testosterone replaced and sham-orchidectomized groups. Testosterone replacement restored the serum testosterone level to that observed in the intact groups as there was no significant difference (p > 0.05) between the testosterone replaced, the intact and the sham orchidectomized groups. A high-salt diet appears to increase the serum level of testosterone. Although not statistically significant, the serum testosterone concentration was consistently and marginally higher in the high-salt-fed groups compared with the normal-salt groups.

Relaxation response to forskolin

Table 2 shows the negative log EC₅₀ of aortic rings relaxation response to forskolin. There was a significant increase (p < 0.05) in the –log EC₅₀ values of the high-salt diet groups compared with the normal-salt diet groups. However, orchidectomy significantly reduced (p < 0.05) the –log EC₅₀ value compared with the value in the intact plus high-salt diet group. Testosterone replacement restored impaired relaxation response to forskolin that was observed in the aorta of rats fed a high-salt diet as there was a significant increase (p < 0.05) in –log EC₅₀ values of the testosterone replaced groups compared with the orchidectomy only group. However, there was no significant difference in the –log EC₅₀ values of both the testosterone replacement group and the intact groups. Figures 1(a)–(d) show the cumulative relaxation response curves of aortic rings from all the groups to forskolin. Figure 2 shows the percentage maximum relaxation response to forskolin in all the groups of rats. Orchidectomy restored the reduced relaxation response to forskolin that was observed in the rats fed a high-salt diet, while concomitant administration of testosterone to the orchidectomized rats re-established the attenuated vasodilatory response to forskolin in the high-salt-fed rats.

Table 2.

Log EC₅₀ of relaxation response to forskolin and diazoxide of aortic rings from the rats.

Groups	Forskolin (mol/liter) n =8	Diazoxide (mol/liter) n = 8
INT + NS	6.99 ± 0.12	6.92 ± 0.13
INT + HS	6.29 ± 0.08^*	6.75 ± 0.14^*
ORCH + NS	7.11 ± 0.10^‡	6.51 ± 0.12^‡
ORCH + HS	^$6.51 ± 0.08^*	^$6.21 ± 0.15^*
ORCH + TES + NS	6.94 ± 0.11	6.84 ± 0.11
ORCH + TES + HS	6.34 ± 0.07^*	6.74 ± 0.13^*
SHAM + NS	6.91 ± 0.10	6.99 ± 0.14
SHAM + HS	6.31 ± 0.07^*	6.36 ± 0.13^*

Data presented as mean ± standard error of the mean.

Significant increase (p < 0.05) compared with corresponding control; ^$significant decrease (p < 0.05) compared with intact and high-salt group; ^‡significant decrease (p < 0.05) compared with control.

EC₅₀, half maximal effective drug concentration; HS, high salt; INT, intact; NS, normal salt; ORCH, orchidectomy; SHAM, sham orchidectomy; TES, testosterone.

Figure 1.

(a) Effect of a high-salt diet. ^*Significant decrease (p < 0.05) in relaxation response compared with intact plus normal diet group. (b) Effect of orchidectomy. ^*Significant decrease (p < 0.05) in relaxation response compared with intact plus normal salt group and orchidectomy plus normal salt group. ^#Significant increase (p < 0.05) in relaxation response compared with intact plus high-salt diet. (c) Effect of testosterone replacement. ^*Significant decrease (p < 0.05) in relaxation response compared with orchidectomy plus normal diet group. ^†Significant decrease (p < 0.05) in relaxation response compared with orchidectomy plus high-salt diet group. (d) Effect of sham orchidectomy on relaxation response of aorta to forskolin in the rats. Data are presented as mean ± standard error of the mean (n = 8). HS, high salt; INT, intact; NS, normal salt; ORCH, orchidectomy; SHAM, sham orchidectomy; TES, testosterone.

Figure 2.

Maximum relaxation response of aortic rings to forskolin in the rats. Data are presented as mean ± standard error of the mean (n = 8). *Significant decrease (p < 0.05) in relaxation compared with control. ^†Significant increase (p < 0.05) in relaxation response compared with orchidectomy and high-salt group. HS, high salt; INT, intact; NS, normal salt; ORCH, orchidectomy; SHAM, sham orchidectomy; TES, testosterone.

Relaxation response to diazoxide

Table 2 shows the negative log EC₅₀ of aortic rings from rats across the groups in response to diazoxide. There was a significant increase (p < 0.05) in the –log EC₅₀, values of the intact plus high-salt diet group compared with the control group. Likewise there was a significant decrease (p < 0.05) in the percentage maximum relaxation response in all the high-salt diet groups compared with the corresponding normal-salt groups (Figure 3). However, there was a significant increase (p < 0.05) in the percentage maximum relaxation response to diazoxide of the orchidectomy plus high-salt diet group compared with that of the intact plus high-salt diet group. Figures 4(a)–(d) show the cumulative relaxation response curves of aortic rings from all the groups to diazoxide. A high-salt diet blunted the relaxation response to diazoxide but orchidectomy abolished the difference in the relaxation response to diazoxide that exists between the normal-fed rats and the high-salt-fed rats. However, testosterone replacement restored the impaired relaxation response to diazoxide in the high-salt diet groups.

Figure 3.

Maximum relaxation response of aortic rings to diazoxide in the rats. Data were presented as mean ± standard error of the mean (n = 8). Significant decrease (^*p < 0.05, ^***p < 0.001) in relaxation response compared with control. ^††Significant increase (p < 0.01) in relaxation response compared with orchidectomy plus high-salt group. HS, high salt; INT, intact; NS, normal salt; ORCH, orchidectomy; SHAM, sham orchidectomy; TES, testosterone.

Figure 4.

(a) Effect of a high-salt diet. ^*Significant decrease (p < 0.05) in relaxation response compared with intact plus normal diet group. (b) Effect of orchidectomy. ^*Significant decrease (p < 0.05) in relaxation compared with intact plus normal-salt diet and orchidectomy plus normal-salt diet. ^#Significant increase (p < 0.05) in relaxation response compared with intact plus high-salt diet. (c) Effect of testosterone replacement. ^*Significant decrease (p < 0.05) in relaxation response compared with orchidectomy plus normal-salt diet. (d) Effect of sham orchidectomy on relaxation response of aorta to diazoxide in the rats. Data are presented as mean ± standard error of the mean (n = 8). HS, high salt; INT, intact; NS, normal salt; ORCH, orchidectomy; SHAM, sham orchidectomy; TES, testosterone.

Discussion

In this study, a high-salt diet impaired relaxation response to forskolin and diazoxide, an adenylyl cyclase activator and a K_ATP channel opener respectively. The impaired vasorelaxation response to forskolin and diazoxide was attenuated by bilateral orchidectomy but concomitant administration of testosterone to orchidectomized rats re-established the impaired relaxation to both adenylyl cyclase and K_ATP activation. Activation of adenylyl cyclase and consequently cAMP production causes vascular smooth muscle relaxation via several downstream mechanisms involving PKA, intracellular calcium ion concentration modulation, opening of potassium channels, among others [Taylor et al. 1999]. Impaired relaxation response to forskolin in rats fed a high-salt diet is consistent with the findings of Sofola and colleagues [Sofola et al. 2003].

The present experiments show that orchidectomy prevented the attenuating effect of a high-salt diet on the aortic rings relaxation response to forskolin, while the effect of orchidectomy was reversed by testosterone replacement. An alternative second-messenger pathway that plays a key role in eliciting relaxation of vascular smooth muscle involves activation of adenylate cyclase, formation of cAMP, and activation of PKA and myosin light-chain kinase within smooth muscle cells [Lamping, 2001]. In general, cAMP-mediated relaxation of vascular smooth muscle does not involve the endothelium [Lamping, 2001]. Likewise forskolin is an endothelium-independent vasodilator. However, some studies have suggested that some ‘typical endothelium-independent’ vasodilators may also release nitric oxide (NO) and activate guanylate cyclase [Taylor et al. 1999]. Relaxation to some classic endothelium-independent agents, including adenosine, prostacyclin, forskolin, and β-receptor agonists, is reduced by inhibitors of NO synthase (NOS) [Taylor et al. 1999], suggesting that relaxation of vascular smooth muscle to these agents is mediated by an interaction between cyclic guanosine monophosphate (cGMP) and cAMP pathways. The mechanisms of NO/cGMP-mediated alterations in cAMP-dependent vascular responses is unclear. Studies demonstrating that vascular responses to forskolin and isoproterenol are attenuated with inhibitors of NOS suggest that these endothelium-independent agents may also release NO [Zhang and Hintze, 2001; Sofola et al. 2003].

Recently, we reported endothelial function impairing the effect of a high-salt diet in male Sprague Dawley rats [Oloyo et al. 2011]. In this study, attenuation of vascular relaxation response to forskolin by a high-salt diet suggests that vascular function impairing the effect of a high-salt diet is endothelial dependent and independent. NO production, which has been reported to be attenuated by a high-salt diet [Zhang and Hintze, 2001], seems to be a common pathway for both forskolin and acetylcholine in eliciting their vasorelaxation. This finding is consistent with our previous suggestion that it is the production or bioavailability of NO that is affected by a high-salt diet rather than the vascular internal mechanism to respond to NO, as evident by the normal relaxation response of aorta from rats fed a high-salt diet to sodium nitroprusside, an exogenous NO donor [Oloyo et al. 2011]. Counteraction by orchidectomy of the effect of a high-salt diet on the relaxation response to forskolin as observed in the present study and that of acetylcholine [Oloyo et al. 2011], and re-establishment of such effects by testosterone replacement suggest testosterone modulates the cGMP/cAMP and NO pathways. The various signal-transduction pathways in vascular tissue that modulate vascular reactivity involve complex interactions and these require further consideration.

Activation of potassium channels causes vascular smooth muscle relaxation via hyperpolarization [Kagota et al. 2002]. Diazoxide is a selective arteriolar vasodilator [Ledoux et al. 2006], which causes vasodilation by hyperpolarizing vascular smooth muscle by opening ATP-dependent K⁺ channels. In the present study, a high-salt diet attenuated the relaxation response of the aorta to diazoxide. Orchidectomy attenuated the impaired aorta relaxation response to the diazoxide effect of a high-salt diet, but testosterone replacement re-established the blunting effect of the high-salt diet on the relaxation response to diazoxide in the aorta. Opening of K_ATP channels is not solely dependent on ATP, as Schwastecher and Schwastecher, and Tasarov and colleagues have reported the involvement of other cytosolic nucleotides including guanosine triphosphate [Schwanstecher and Schwanstecher, 2002; Tasarov et al. 2004]. Similarly, the stimulatory effect of PKA, a cAMP-dependent protein kinase, on K_ATP channels has been reported [Lin et al. 2000; Tammaro, 2009].

A high-salt diet has been reported to attenuate cAMP [Sofola et al. 2003], as well as NO/soluble guanylyl cyclase/cGMP pathway mediated vasorelaxation [Kagota et al. 2002]. In this study, attenuation of the aorta relaxation response to diazoxide by a high-salt diet could be due to the effect of a high-salt diet on the vascular smooth muscle nucleotides and their respective pathways of eliciting vasorelaxation. This finding is consistent with the attenuation by a high-salt diet of the aorta relaxation response to forskolin, which elicits its vasorelaxing effect via the cAMP pathway. Improvement by orchidectomy of the impaired vasorelaxation response of the aorta from animals fed a high-salt diet implicates testosterone in the increase in vascular tone observed in male Sprague Dawley rats and suggests that part of such an effect is mediated through the cAMP pathway and K_ATP channel activation.

The slight increase in the plasma concentration of testosterone in rats fed a high-salt diet is quite interesting because, like a high-salt diet, the effect of chronic exposure to testosterone is an increase in vascular tone [Gonzales et al. 2004]. Although, to the best of our knowledge, there is no literature on the effect of a high-salt diet on the serum level of testosterone, ancient Greek mythology documents the use of salt by wives to increase their husband’s libido [Engraving, 1557, Bibliothèque Nationale (Estampes), Paris]. Likewise, salt has been reported to increase fertility and enhance reproductive functions [Moinier and Drueke, 2008]. Experimental findings have also shown that aldosterone stimulates testosterone production from the Leydig cells [Ge et al. 2005]. In addition, flutamide, an androgen receptor blocker, increases the serum level of aldosterone in orchidectomized rats [Hoffman et al. 2012]. These findings suggest an interaction between salt intake, its main regulatory hormone (aldosterone) and testosterone. This is not impossible considering the fact that aldosterone and testosterone are steroidal hormones which, apart from sharing the same precursor, are both formed through steroidogenesis. It is possible that testosterone potentiates the vascular effect of a high-salt diet, which may explain the greater susceptibility of male rats to a high-salt diet compared with female rats [Hinojosa-Laborde et al. 2004].

Findings from this study suggest that testosterone enhances the impairment of vascular function by a high-salt diet. Considering the role of a high-salt diet in cardiovascular diseases and death, the vascular effect of testosterone may account for the higher susceptibility of men to cardiovascular diseases and death compared with age-matched premenopausal women.

Conclusion

Modulation of the cAMP pathway and potassium channel activity appears to contribute to the mechanisms by which a high-salt diet increases vascular tone. This effect was counteracted by orchidectomy in male Sprague Dawley rats, suggesting the possible involvement of testosterone in the vascular response.

Footnotes

Funding

This work was supported by University of Lagos Central Research Committee (CRC) research grant 2007/14. A.K. Oloyo is a beneficiary of the INSA JRD – TATA Fellowship from the Center for Cooperation in Science and Technology among Developing Societies (CCSTDS) of the Federal Government of India.

Conflict of interest statement

The authors declare no conflicts of interest in preparing this article.

References

Chappell

Westwood

Yamaleyeva

(2008) Differential effects of sex steroids in young and aged female mren2.lewis rats: a model of estrogen and salt-sensitive hypertension. Gend Med 5: S65–S75.

Cox

Cohen

(1997) Influence of gender on vasomotor effects of oxidized low-density lipoprotein in porcine coronary arteries. Am J Physiol Heart Circ Physiol 272: H2577–H2583.

Domoki

Kis

Nagy

Farkas

Busija

Bari

(2005) Diazoxide preserves hypercapnia-induced arteriolar vasodilation after global cerebral ischemia in piglets. Am J Physiol Heart Circ Physiol 289: H368–H373.

Dong

Sottas

Latif

Morris

Hardy

(2005) Stimulation of testosterone production in rat Leydig cells by aldosterone is mineralocorticoid receptor mediated. Mol Cell Endocrinology 243: 35–42.

Gonzales

Krause

Duckles

(2004) Testosterone suppresses endothelium-dependent dilation of rat middle cerebral arteries Am J Physiol Heart Circ Physiol 286: H552–H560.

Hinojosa-Laborde

Craig

Zheng

Haywood

Sandberg

(2004) Ovariectomy augments hypertension in aging female Dahl salt sensitive rats. Hypertension 44: 405–409.

Hoffman

Michaelis

Gotz

Bartel

Kienitz

Quinkler

(2012) Flutamide increases aldosterone levels in gonadectomised male but not female Wistar rats. Am J Hypertens 25: 697–703.

Kagota

Tamashiro

Yamaguchi

Nakamur

Kunitomo

(2002) High salt intake impairs vascular nitric oxide/cyclic guanosine monophosphate system in spontaneously hypertensive rats. JPET 302: 344–351.

Kneale

Chowienczyk

Brett

Coltart

Ritter

(2000) Gender differences in sensitivity to adrenergic agonists of forearm resistance vasculature. J Am Coll Cardiol 6: 1233–1238.

10.

Lamping

(2001) Interactions between NO and cAMP in the regulation of vascular tone. Arterioscler Thromb Vasc Biol 21: 729–730.

11.

Ledoux

Werner

Brayden

Nelson

(2006) Calcium-activated potassium channels and the regulation of vascular tone. Physiology 21: 69–79.

12.

Lin

Jan

(2000) Regulation of ATP-sensitive potassium channel functions by protein kinase A-mediated phosphorylation in transfectedHEK293 cells. EMBO J 19: 942–955.

13.

Liu

Death

Handelsman

(2003) Androgens and cardiovascular disease. Endocrine Rev 24: 313–340.

14.

Marcus

Durnford

(1986) A simple enzyme-linked immunosorbent assay for testosterone. Steroids 46: 975–986.

15.

Meneton

Jeunemaitre

de Wardener

MacGregor

(2005) Links between dietary salt intake, renal salt handling, blood pressure, and cardiovascular diseases. Physiol Rev 85: 679–715.

16.

Miller

(2000) An overview of the potassium channel family. Genome Biol 1: 0004.1–0004.5.

17.

Moinier

Drueke

(2008) Aphrodite, sex and salt – from butterfly to man. Nephrol Dial Transplant 23: 2154–2161.

18.

Newgreen

Bray

McHarg

Weston

Duty

Brown

. (1990) The action of diazoxide and minoxidil sulphate on rat blood vessels: a comparison with cromakalim. Br J Pharmacol 100: 605–613.

19.

Nurkiewicz

Boegehold

(2007) High salt intake reduces endothelium-dependent dilation of mouse arterioles via superoxide anion generated from nitric oxide synthase. Am J Physiol Regulatory Integrative Comp Physiol 292: R1550–R1556.

20.

Oloyo

Sofola

Anigbogu

(2011) Orchidectomy attenuates impaired endothelial effects of a high salt diet in Sprague-Dawley rats. Can J Physiol Pharmacol 89: 295–304.

21.

Pasdois

Beauvoit

Tariosse

Vinassa

Bonoron-Adèle

Dos Santos

(2008) Effect of diazoxide on flavoprotein oxidation and reactive oxygen species generation during ischemia-reperfusion: a study on Langendorff-perfused rat hearts using optic fibers. Am J Physiol Heart Circ Physiol 294: H2088–H2097.

22.

Pojoga

Yao

Sinha

Ross

Lin

Raffetto

(2008) Effect of dietary sodium on vasoconstriction and eNOS-mediated vascular relaxation in caveolin-1-deficient mice. Am J Physiol Heart Circ Physiol 294: H1258–H1265.

23.

Polleselloa

Mehazaab

(2004) ATP-dependent potassium channels as a key target for the treatment of myocardial and vascular dysfunction. Curr Opin Crit Care 10: 436–441.

24.

Sader

Celermajer

(2000) Endothelial functions, vascular reactivity and gender differences in the cardiovascular system. Cardiovasc Res 53: 597–607.

25.

Sandhiya

Dkhar

(2009) Potassium channels in health, disease & development of channel modulators. Indian J Med Res 129: 223–232.

26.

Schmidlin

Forman

Sebastian

Morris

RC.

Jr (2007) What initiates pressor effect of salt in salt sensitive Humans? Observation in normotensive blacks. Hypertension 49: 1032–1039.

27.

Schwanstecher

(2002) Nucleotide sensitivity of pancreatic ATP-sensitive potassium channels and type 2 diabetes. Diabetes 51(Suppl. 3): S358–S362.

28.

Sofola

Yakubu

Igbo

Newaz

Oyekan

(2003) High salt diet modulates cAMP and nitric oxide-mediated relaxation response to isoprotenol in the rat aorta. Eur J Pharmacol 472: 241–247.

29.

Tammaro

(2009) Vascular KATP channels: dephosphorylation and deactivation. Br J Pharmacol 157: 551–553.

30.

Tarasov

Dusonchet

Ashcroft

(2004) Metabolic regulation of the pancreatic β-cell ATP-sensitive K⁺ channel. A Pas de Deux. Diabetes 53(Suppl. 3): S113–S122.

31.

Taylor

McMahon

Gardner

Benoit

(1999) Cyclic nucleotides and vasoconstrictor function: physiological and pathophysiological considerations. Pathophysiology 5: 233–245.

32.

Thompson

Khalil

(2003) Gender differences in the regulation of vascular tone. Clin Exp Pharmacol Physiol 30: 90–105.

33.

Xia

Khalil

(2010) Sex-related decrease in [Ca²⁺]i signaling and Ca²⁺-dependent contraction in inferior vena cava of female rat. Am J Physiol Regul Integr Comp Physiol 298: R15–R24.

34.

Zhang

Hintze

(2001) cAMP signal transduction cascade, a novel pathway for the regulation of endothelial nitric oxide production in coronary blood vessels. Arterioscler Thromb Vasc Biol 21: 797–803.

35.

Zhu

Huang

Lombard

(2007) Effect of high-salt diet on vascular relaxation and oxidative stress in mesenteric resistance arteries. J Vasc Res 44: 382–390.

36.

Zhu

Friesema

Huang

Roman

Lomband

(2005) Salt–induced ANG II suppression impairs the response of cerebral artery smooth muscle cells to prostacyclin. Am J Physiol Heart Circ Physiol 288: H908–H913.