Testosterone relaxes abdominal aorta in male Sprague–Dawley rats by opening potassium (K+) channel and blockade of calcium (Ca2+) channel

Pathophysiology 18 (2011) 247–253

Testosterone relaxes abdominal aorta in male Sprague–Dawley rats by opening potassium (K+) channel and blockade of calcium (Ca2+) channel Ahmed Kolade Oloyo a,∗ , Olusoga A. Sofola a , Renuka R. Nair b , V.S. Harikrishnan c , Adelaide C. Fernandez c b

a Department of Physiology, College of Medicine, University of Lagos, Idi – Araba Lagos, Nigeria Division of Cellular and Molecular Cardiology (DCMC), Sree Chitra Tirunal Institute for Medical Sciences (SCTIMST), Medical College, Trivandrum, Kerala, India c Department of Laboratory Animal Sciences (DLAS), Biomedical Technology (BMT) Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST), Poojapurah, Trivandrum, Kerala, India

Received 12 July 2010; received in revised form 22 February 2011; accepted 22 February 2011

Abstract Aim: To investigate the direct effect of testosterone and its precursor/derivative dehydroepiandrosterone (DHEA) on isolated rat abdominal aortic rings. Materials and methods: 3 mm abdominal aortic rings that were obtained from 3 months old male Sprague–Dawley rats were suspended in organ baths containing Hepes buffered PSS bubbled with 100% oxygen. Relaxation response to testosterone and DHEA was studied in noradrenalin pre-contracted rings. The role of aromatase and androgen receptor was assessed by inhibition using aminogluthetemide and blockade using flutamide respectively. Relaxation responses of the rings to testosterone in the presence of l-NAME, indomethacin, barium chloride, apamin, charybdotoxin, iberiotoxin, and nifedipine were also determined. Results: Both aromatase inhibition and androgen receptor blockade did not block the relaxing effect of testosterone on rings from rat abdominal aorta. Also there was no significant difference between testosterone relaxation response in the presence or absence of l-NAME and indomethacin. However 3 ␮M, BaCl2 almost completely abolished the aortic ring relaxation response to testosterone while 1 ␮M, nifedipine potentiated the vasorelaxing effect of testosterone. Conclusion: Testosterone relaxes abdominal aorta directly via a non-genomic pathway which is independent of endothelial derived vasoactive substances, but involves activation of inward rectifying potassium channel (KIR ) and blockade of l-type calcium channel. © 2011 Elsevier Ireland Ltd. All rights reserved. Keywords: Testosterone; Non-genomic; Dehydroepiandrosterone; Vascular relaxation; Potassium channel; Calcium channel

1. Introduction Many studies have reported that there is pronounced sexual dimorphism in many cardiovascular diseases, most notably hypertension and coronary artery diseases [1], as it has been reported that there is a greater incidence of hypertension and coronary artery disease in men and post-menopausal women when compared with pre-menopausal women [2]. Higher male susceptibility to cardiovascular disease may be due to genetic, hormonal or lifestyle factors or a combination of mechanisms. Out of these factors, hormonal effects are the most tractable for practical therapeutic purposes, considering the various reproductive steroids that are available [1]. Fur∗

Corresponding author. Tel.: +234 8028363152. E-mail address: [email protected] (A.K. Oloyo).

0928-4680/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2011.02.004

thermore it has been reported that testosterone injection has a relieving effect on patient with angina pectoris [3,4] is beneficial to men with coronary artery disease [5] or congestive heart failure [6,7]. This suggests that testosterone may have an effect on vascular function. Although the classical pathway of androgen action involves steroid binding to the androgen receptor (AR) [8,9], there are now considerable evidence for rapid, non-genomic effect of steroids including androgens [10]. Non-genomic effects of androgen usually involve the rapid induction of conventional second-messenger signal transduction pathways, such as increases in cytosolic calcium and activation of protein kinase A (PKA), protein kinase C (PKC), and mitogen activated protein kinase (MAPK), leading to diverse cellular effects which includes smooth muscle relaxation [9]. No membrane AR has been characterized, but preliminary evidence of a low affinity microsomal

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membrane binding site for alkylated androgens [11] and an endothelial cell plasma membrane dehydropiandrostenodione (DHEA) binding site [12] still require functional proof of specific receptor status. Also a plasma membrane SHBG membranes and initiating intracellular cAMP signaling has been described in humans [13]. The sex hormone binding globulin (SHBG) receptor remains to be fully characterized, and it is not clear whether it has any physiological role in species like rodents that lack circulating SHBG. Sex steroids (oestrogen, progesterone and testosterone) receptors have been identified in blood vessels of human and other mammals and have been localized in the plasmalemma, cytosol and nuclear compartment of various cells including the endothelium and the vascular smooth muscle [2,14]. Most of the non-genomic effects of androgen on the vascular tone have been described in rabbit coronary arteries and aorta, where testosterone relaxes these vessels [15,16], as well as in rat thoracic aorta [17]. Recently, Kouloumenta et al. [18] reported an in vitro relaxing effect of testosterone on rabbit airway smooth muscle while the vasorelaxing effect of testosterone on human radial artery [19] and pulmonary artery [20] have also been reported. Differences exist in the responses of vascular cells from different parts and different species to vasoactive substances, for example, rabbit aorta is more responsive to norepinepherine than its branches, a fact that has been ascribed to variations in response to extracellular calcium ions along the aorta [21,22], while rat thoracic aorta and mesenteric artery exhibit differential responses to norepinepherine and serotonin [23]. Therefore this study was designed to investigate the direct effect of testosterone and its precursor/derivative dehydroepiandrosterone (DHEA) on isolated abdominal aortic rings from male Sprague–Dawley rats in the presence or absence of flutamide, aminogluthetemide, l-nitro arginine metyl ester (l-NAME), indomethacin, barium chloride (BaCl2 ) tertiapin-Q, iberiotoxin, charybdotoxin, apamin and nifedipine.

2. Materials and methods Experimental protocol was approved by the Animal Research and Ethics Committee of the Biomedical Wing Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST), Trivandrum, Kerala, India. 2.1. Tissue preparation Ten inbred adult male Sprague–Dawley rats obtained from Department of Laboratory Animal Sciences (DLAS), BioMedical and Technology (BMT) wing Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST) Kerala, India, weighing 250–300 g were killed 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 PSS solution. 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-shaped 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 from ADInstruments, 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 Hepes buffer was between 7.35 and 7.40, and all baths used simultaneously had a parallel connection to the source of Hepes buffer. The composition of the solution in mmol/L was NaCl 133, KCl 3.6, CaCl2 1.8, MgCl2 ·6H2 O 1.2, glucose 16, Hepes 30, and KH2 PO4 1.18. 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 each ring was subjected to a sub-maximal dose (0.1 ␮M) of noradrenalin at 30 min interval. Isometric tension was then measured using the top force transducer which was connected to a Powerlab 2/25 recorder (ADInstruments, Australia). 2.2. Experimental procedure At the end of the 90 min stabilization period, cumulative concentration–response curves to testosterone propionate and dehydroepiandrosterone (DHEA) (0.1–100 ␮M) were obtained in aortic rings that were pre-contracted with 0.1 ␮M noradrenalin. The role of aromatase (CPY19), an enzyme that converts testosterone to estradiol in the peripheral tissues, as well as androgen receptor was assessed by inhibition using aminogluthetemide (5 ␮M) and blockade using flutamide (10 ␮M) respectively. The involvement of endothelial vasoactive substances on the effect of testosterone was assessed by incubating some aortic rings with l-NAME (100 ␮M) an endothelial nitric oxide synthase (eNOS) inhibitor, and indomethacin (10 ␮M) a cyclooxygenase-2 (Cox-2) inhibitor. Similarly, concentration–response curves to testosterone propionate were obtained after incubation of aortic rings in some potassium channel blockers; barium chloride (3 ␮M), a non-selective inward rectifying K+ channel (KIR ) blocker, tertiapin-Q (100 nM), a selective inward rectifier K+ channel (Kir 1.1, Kir 3.x subfamilies of KIR ) blocker, apamin (1 ␮M), charybdotoxin (1 ␮M), and iberiotoxin (25 nM) which are, selective small, intermediate and large conductance calciumactivated K+ channel blockers respectively. On the other hand, the role of voltage dependent calcium channel (VDCC) on the effect of testosterone was assessed by incubating some aortic rings in nifedipine (1 ␮M), a conventional ltype calcium channel blocker. Finally, in order to evaluate if calcium channel blockade augments the relaxing effect of testosterone on the aortic rings, we compared its calcium channel blocking potential with that of nifedipine, a conventional l-type calcium channel blocker. Cumulative concentration–response curves to nifedipine (0.1–100 ␮M), after incubation of the aortic ring in 1 ␮M testosterone for 30 min, were then obtained.

A.K. Oloyo et al. / Pathophysiology 18 (2011) 247–253 100

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0 TESTOSTERONE DHEA

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Fig. 1. Relaxing effect of testosterone propionate (0.1, 1, 10, and 100 ␮M) on abdominal aorta of male Sprague–Dawley rats. Data are expressed as percentage relaxation of contraction induced by noradrenalin (0.1 ␮M) (mean ± S.E.M., n = 6–10). Control indicates time-matched (5 min) ethanol solvent controls. *Significant increase in comparison with control, P < 0.05. **Significant increase in comparison with control, P < 0.01. ***Significant increase in comparison with control P < 0.001.

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Log Concentration of Testosterone and DHEA (mol/L) Fig. 2. Relaxation effect of dehydroepiandrosterone (DHEA) on abdominal aorta of male Sprague–Dawley rats. Values are expressed as means ± S.E.M. (n = 10).

3. Results 2.3. Drugs and solutions

2.4. Data analysis Relaxation responses to testosterone propionate and DHEA were expressed as percentage of the active tone achieved with 0.1 ␮M noradrenalin. Relaxation responses to testosterone in the presence of different modulators were compared with that of testosterone alone. Half-maximal effective drug concentration (EC50 ) was calculated for each concentration response curve [24], using Graphpad Prism statistical software. The pEC50 was calculated as the negative logarithm to base 10 of the EC50. 2.5. Statistical analysis The collected data was expressed as mean ± S.E.M. and were analyzed using one-way analysis of variance (ANOVA) with Student–Newman–Keuls test post hoc to identify differences between individual means.

Administration of cumulative pharmacological doses of testosterone resulted in concentration-dependent relaxation of active tone in rat abdominal aorta, as shown in Fig. 1. There was a significant increase (P < 0.05) in the percentage relaxation response of the aortic rings to 1, 10 and 100 ␮M testosterone when compared with time-matched ethanol solvent control. As shown in Fig. 2, there was no significant difference in the relaxation response to DHEA when compared with that of testosterone. The relaxation response to testosterone was not significantly modified by incubation of aortic rings with aminogluthetemide, an aromatase inhibitor or flutamide an androgen receptor blocker. (Fig. 3). Also there was no significant difference between testosterone induced relaxation response in the presence or absence of 100 ␮M l0 Control Aminoglut (5µM) & Fluta (10 µM)

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Testosterone propionate, dehydroepiandrosterone (DHEA), aminogluthetemide, and flutamide were dissolved and diluted in ethanol, while nifedipine was dissolved in 50% ethanol. Charybdotoxin and iberiotoxin were dissolved in 100 mM of sodium chloride while apamin was dissolved in 5% acetic acid and then diluted in distilled water. Noradrenalin, tertipin-Q, l-NAME and barium chloride were dissolved and diluted in distilled water while indomethacin was dissolved in 2.5% sodium carbonate and diluted in distilled water. All drugs and chemicals used were obtained from Sigma–Aldrich Chemicals (Bangalore, India).

40

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Log Concentration of Testosterone (mol/L) Fig. 3. Concentration–response curves to testosterone in rat abdominal aorta following incubation in 5 ␮M aminogluthetemide (Aminoglut) and 10 ␮M flutamide (Fluta). Values are expressed as means ± S.EM. (n = 6).

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Fig. 4. Relaxation response to testosterone before and after addition of lNAME or indomethacin (Indo) in rat abdominal aorta. Values are expressed as means ± S.E.M. (n = 6).

0

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Control BaCl 2 (3 µ M)

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Fig. 6. Effect of preincubation of the rat abdominal aorta with 1 ␮M nifedipine on relaxation response to testosterone. Values are expressed as means ± S.E.M. (n = 6). **Significant increase in relaxation respose when compared with control (P < 0.01).

On the other hand, there was a significant increase (P < 0.01) in the relaxation response to testosterone (EC50 5.84 ± 0.11) mol/L with l-type calcium channel blockade by 10−6 M nifedipine when compared with relaxation response to testosterone alone (EC50 4.95 ± 0.07) mol/L, (Fig. 6). Fig. 7 shows the comparison in the calcium channel block0 TEST + NIFE (1µM) NIFE + TES (1µM)

20

% Relaxation

NAME, and 10 ␮M indomethacin (Fig. 4). However as shown in Fig. 5, 3 ␮M BaCl2 , a non-selective inward rectifier potassium channel (KIR ) blocker, almost completely abolished the aortic ring relaxation response to testosterone. There was a significant decrease (P < 0.01) in the relaxation response (EC50 5.22 ± 0.12) mol/L of abdominal aorta to testosterone after 30 min incubation with barium chloride when compared with control (EC50 4.95 ± 0.07) mol/L. Blockade of the small, intermediate and large conductance calcium-activated potassium channels and the subfamilies of inward rectifier potassium channel (Kir 1.1, Kir 3.x) by apamin, charybdotoxin, iberiotoxin and tertiapin-Q respectively had no effect on the relaxation response of the abdominal aorta to testosterone (data not included). At the concentrations employed, these drugs are specific for the respective potassium channels [15,25,26].

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Log Concentration of Testosterone and Nifedipine (mol/L) 100 -8

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Log Concentration of Testosterone (mol/L) Fig. 5. Effect of preincubation of the rat abdominal aorta with 3 ␮M barium chloride on relaxation response to testosterone. Values are expressed as means ± S.E.M. (n = 6). **Significant decrease in relaxation response when compared with control (P < 0.01).

Fig. 7. Comparison of the calcium channel blocking potential of testosterone with l-type calcium channel blocker nifedipine in rat abdominal aorta. Values are expressed as means ± S.E.M. (n = 6). **Significant decrease in relaxation when compared with NIFE + TEST (P < 0.01). (TEST, testosterone, NIFE, nifedipine. TEST + NIFE: aortic ring relaxation response to nifedipine after 30 min incubation in 1 ␮M testosterone, NIFE + TEST: aortic ring relaxation response to testosterone after 30 min incubation in 1 ␮M nifedipine).

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ade activity of both nifedipine and testosterone. There was a significant difference (P < 0.01) in the relaxation response to testosterone (EC50 5.84 ± 0.11) mol/L in the presence of nifedipine when compared with the relaxation response to nifedipine (EC50 5.24 ± 0.19) mol/L in the presence of testosterone. Cumulative doses of nifedipine induced a less concentration dependent relaxation in the abdominal aorta when compared with that of testosterone in the presence of nifedipine. (1) Abdominal aorta relaxation response to testosterone and time-matched control (ethanol) (Fig. 1). (2) Abdominal aorta relaxation response to testosterone and dehydroepiandrosterone (DHEA) (Fig. 2). (3) Abdominal aorta relaxation response to testosterone in the presence of aminogluthetemide and flutamide (Fig. 3). (4) Abdominal aorta relaxation response to testosterone in the presence of l-NAME and indomethacin (Fig. 4). (5) Abdominal aorta relaxation response to testosterone in the presence of barium chloride (Fig. 5). (6) Abdominal aorta relaxation response to testosterone in the presence of nifedipine (Fig. 6). (7) Comparison of calcium channel bloking effect of testosterone and nifedipine (Fig. 7).

4. Discussion Abdominal aortic rings were used in the present experiment. This was because there are mechanistic differences in response to vasoactive substances between thoracic aorta and abdominal aorta. For example thoracic aorta has mainly EDRF activity and little or no EDHF while abdominal aorta on the other hand has been shown to exhibit some EDHF activity (Sofola and Oyekan, personal communication). The classical pathway of androgen action involves steroid binding to the androgen receptor (AR), a ligand activated transcription factor, and single copy member of the nuclear receptor super family, acting on the genome [8]. There is now considerable evidence for a rapid, non-genomic effect of steroids, including androgens [10]. Non-genomic steroid action is distinguished from genomic effect by the rapid onset of its effect (seconds to minutes) that is faster than genomic mechanisms. Also such effects are insensitive to inhibition of RNA and protein synthesis and can be produced by steroids that have been rendered impermeable to the plasma membrane, thus unable to access the nucleus. Nontranscriptional effects of androgens are also not usually blocked by classical antagonists due to their different steroid specificities from classical cognate nuclear receptor [27]. A key issue in the biological effects of testosterone is its conversion to bioactive metabolites. Although only a small fraction (<5%) of testosterone output undergoes such transformation usually in local tissues, conversion both amplifies and diversifies testosterone action. Conversion to its 5␣-

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reduced metabolite, dihydrotestosterone (DHT) by type 1 or type 2, 5␣-reductase amplifies testosterone action because DHT has higher molar potency due to its more avid binding affinity and slower dissociation rate from the androgen receptor (AR) [28]. Conversion of testosterone to estradiol by the enzyme aromatase (CYPI9) diversifies androgen action by activating oestrogen receptors (ER) [29–32]. The result from this study shows that neither flutamide, a classical androgen receptor blocker nor aminogluthetemide, an aromatase inhibitor blocked the relaxation response of the aortic rings to testosterone. This suggests that acute administration of testosterone propionate causes relaxation in the abdominal aorta via a pathway that is independent of the nuclear receptors activation, nor through peripheral conversion to estradiol. Non-genomic androgen effects characteristically involve the rapid induction of conventional second-messenger signal transduction cascades, such as increases in cytosolic calcium and activation of PKA, PKC, and MAPK, leading to diverse cellular effects including smooth muscle relaxation, neuromuscular and junctional signal transmission and neuronal plasticity [9]. Modulations of ion channel activities are part of the mechanisms through which the effects of second messenger activation are observed. Activation of potassium channels both in the endothelium and vascular smooth muscle causes hyperpolarization which leads to vascular smooth muscle relaxation [33,34]. Mechanisms involving potassium channel activation have been implicated in vascular smooth muscle contraction. For instance Na+ K+ 2Cl− cotransport (NKCC) activity has been demonstrated to mediate cell-volume induced contraction in VSM contraction [35], myogenic tone [36], and excitation–contraction coupling in resistance mesenteric arteries [37,38]. NKCC was reported to be sensitive to millimolar concentration (5 mmol/L) of BaCl2 [39] and at similar range it is also a nonspecific blocker of K+ channel [15]. BaCl2 at the concentration used in this present study is a nonselective KIR blocker [25]. In the present study, attenuation of vascular relaxation response to testosterone propionate in the presence of barium chloride, a nonselective KIR blocker, implicates KIR channel activation in the vasorelaxing effect of acute administration of testosterone propionate. Unlike potassium channels, calcium channels activation has opposing effect in the endothelium and the vascular smooth muscle cells (VSMCs). In the endothelial cells calcium channel activation eventually leads to vascular smooth muscle relaxation, while in the VSMCs, activation of calcium channel causes contraction. In this present study, it was observed that the endothelium plays little or no role in the relaxing effect of testosterone propionate on the vascular smooth muscle. This is so because inhibition of endothelial nitric oxide synthase (eNOS) by l-NAME or prostacyclin by indomethacin had no effect on the relaxation response of the aorta to testosterone. Endothelial K+ channels have been widely implicated in endothelium-dependent vasodilatation. It was thought that endothelial cell hyperpolarization, via the opening of K+ channels, would facilitate Ca2+ influx in these cells by increasing the driving force

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for Ca2+ [40,41], and in this way enhance production of the “classical” endothelium-dependent vasorelaxants NO and PGI2, which rely on increase in cytoplasmic free Ca2+ . However the discovery of additional vasodilator phenomenon of endothelium-derived hyperpolarizing factor (EDHF) showed that in addition to causing increased cytoplasmic Ca2+ , endothelial K+ channels inadvertently cause adjacent smooth muscle hyperpolarization. This arises from activation of small and intermediate calcium activated K+ channels in endothelial cells, resulting in endothelial hyperpolarization which spreads through myoendothelial junctions to result in the EDHF-attributed hyperpolarization and relaxation of the smooth muscle [42]. Small, intermediate and large conductance Ca2+ -activated K+ channels have been implicated in EDHF activity, while inward rectifier K+ channels (KIR ) and Na+ /K+ ATPase have been suggested by some studies [43]. IKCa and SKCa channels occur in abundance in endothelial cells and their activation results in EDHF-like hyperpolarization of these cells. K+ efflux from endothelial cells via small and intermediate calcium activated K+ channels (SKCa and IKCa respectively), activates inward rectifier K+ channels (KIR ) and Na+ /K+ ATPase on the smooth muscle cells [44]. In this present study, blockade by apamin, charybdotoxin and iberiotoxin of small, intermediate and large conductance calcium-activated potassium channel respectively, which are found mainly on the endothelium had no effect on the vasorelaxing effect of testosterone. This is consistent with the earlier findings that reported that endothelium may not be involved in the relaxation effect of testosterone [17]. In the vascular wall, inward rectifier potassium channels (KIR ) channels are expressed in both the endothelial and smooth muscle cells [45,46]. Kir 1.1 and Kir 3.x subfamilies of KIR are expressed more in the smooth muscle of autoregulatory vascular bed such as cerebral and coronary arteries [45,47], while their expression in the general circulation increases with decreasing arterial diameter [48,49]. KIR channel conducts potassium current more readily in the cells than out of the cells over a wide range of potentials. Activation of KIR results in hyperpolarization and thus relaxation of smooth muscle [42]. In this present study, tertiapin-Q a selective blocker of Kir 1.1 and Kir 3.x subfamilies of inward rectifier potassium channel failed to prevent the relaxing effect of testosterone on the abdominal aorta. As explained above, it could be that there is no significant expression of Kir 1.1 and Kir 3.x in the abdominal aorta cells, or activation of these subfamilies of KIR is not involved in the pathway through which testosterone induces relaxation in the male Sprague–Dawley rats abdominal aorta. In this study, nifedipine, a conventional l-type calcium channel blocker, was found to augment the relaxation response of testosterone on the aorta, suggesting that calcium channel blockade could be another mechanism by which testosterone relaxes vascular smooth muscle in the abdominal aorta. Calcium channel blocking effect of testosterone could be another reason why blockade of the three types of calcium activated potassium channel failed to have any effect on the vasorelaxing activities of testosterone. It

could be that calcium channel blocking effect of testosterone prevented the intracellular/cytosolic concentration of calcium ions from reaching the required level to activate these calcium ion dependent potassium channels; an important step in their activation [41]. In this present study, the fact that testosterone causes further relaxation even after incubation with nifedipine while nifedipine cannot elicit further relaxation after incubation in testosterone suggests that at the same concentration, testosterone blocks more l-Ca2+ channels than nifedipine. This result may suggest that testosterone is a more potent l-Ca2+ channel blocker when compared with nifedipine a conventional l-type calcium channel blocker. The results of this present study therefore suggest that testosterone propionate relaxes abdominal aorta from male Sprague–Dawley rats directly via a non-genomic pathway which is virtually not dependent on the endothelial vasoactive substances. Inward rectifier potassium channel activation and l-type calcium channel blockade appear to be involved in the pathway by which testosterone induces its vasorelaxing activity in the abdominal aorta of male Sprague–Dawley rats.

Acknowledgements This study was funded by UNILAG CRC research grant 2007/14. A.K. Oloyo, is a beneficiary of INSA JRD–TATA Fellowship from the Center for Cooperation in Science and Technology among Developing Societies (CCSTDS) of the Federal Government of India.

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