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Relaxation of rabbit cavernous smooth muscle to 17-estradiol: a non-genomic, NO-independent mechanism

Sae-Chul Kim1, Kyung-Kun Seo1, Soon-Chul Myung1, Moo Yeol Lee2

1Department of Urology, 2Department of Physiology, College of Medicine, Chung-Ang University, Seoul 140-757, Korea

Asian J Androl 2004 Jun; 6: 127-131
Keywords: 17-estradiol; cavernous smooth muscles; Maxi-K channel; relaxation
Abstract

Aim: To investigate whether estrogen was involved in relaxation of rabbit cavernous smooth muscle. Methods: Relaxation response of the rabbit cavernous smooth muscles to 17-estradiol (0.3, 3, 30 and 300 nmol/L) were observed in vitro. The response of the muscle strips to estrogen after incubation with either actinomycin D (10 mol/L) or L-NAME (10 mol/L) were also evaluated. Inside-out mode of patch clamp in a single smooth muscle cell was applied to investigate the Maxi-K channel activities. Results: Estrogen caused a dose-dependent relaxation of the strips precontracted with norepinephrine. The maximal response was noted about 10 minutes after treatment. The estrogen-induced relaxation was prevented by neither actinomycin D nor L-NAME, suggesting that the response was not mediated by gene transcription or nitric oxide (NO). Application of 17-estradiol increased the Maxi-K channel activities. Conclusion: 17-estradiol may be involved in relaxation of rabbit cavernous smooth muscles via a non-genomic and NO independent mechanism. 17-estradiol stimulates Maxi-K channel of the rabbit cavernous myocyte.

1 Introduction

The daily production rate of estrogens in men is comparable to that by a postmenopausal woman. In adult men the molar ratio of plasma testosterone to estrogen usually ranges at about 300 : 1. It is now becoming more and more evident that estrogens play an important role in male reproduction, including the masculinization of the brain for the development and the maintenance of sexual behavior in men [1, 2].

Estrogen treatment enhances the endothelium-dependent relaxation in femoral, coronary and cerebral arteries [3-5], as well as the endothelium-independent vasodilatory responses to estrogen [6, 7]. Depending on the cell type, the distribution and relative expression levels of estrogen receptors (ER) vary and ER subtype, either ER or ER, can be predominantly expressed in estrogen targeted tissues. The ER and ER were also expressed in penile cavernous tissues [8]. It is well known that testosterone is involved in local reactions related with penile erection. However, the role of estrogens in penile erection has not been clarified so far, while hyperesterogenemia is known to lead to erectile dysfunction [9]. This study was aimed to determine whether estrogen is involved in the relaxation response of cavernous smooth muscle.

2 Materials and methods

 

2.1 Preparation of rabbit cavernous strips

A total of 20 New Zealand white rabbits (2.5 - 3 kg) were killed and the entire penis was removed. The cavernous tissue was carefully dissected free from the tunica albuginea. The excised cavernous tissues were immediately placed in 100 % oxygen-saturated Tyrode's solution for 30 minutes. Strips of cavernous smooth muscles were trimmed to a size of 0.2 cm0.2 cm1.0 cm and mounted in a 30 mL organ chamber filled with HEPES buffered Tyrode's solution bubbled with 100 % O2 and maintained at 37 , pH 7.4. The physiologic solution was exchanged at 30 minute intervals. The strip was connected by a silk tie to a force transducer (52-9545, Harvard, UK) and polygraph (50-8630, Harvard, UK) to record the isometric tension. The passive tension was adjusted to 1.5 g over a 30 minute equilibration period.

2.2 Chemicals and reagents

Acetylcholine chloride, norepinephrine (NE), HEPES, Nw-nitro-L-arginine methyl ester (L-NAME), 17-estradiol (17-E2), actinomycin D and iberiotoxin were purchased from Sigma Chemical Co. (USA).

The composition of the HEPES-buffered physiological solution was as follows: 140 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2, 5 mmol/L HEPES and 11 mmol/L glucose and titrated to pH 7.4 with 1 mol/L NaOH.

2.3 Pharmacological responses

After smooth muscle strips reached a steady state contraction to ED50 NE (10-9-10-4 mol/L), doses of 17-E2 were added to the tissue bath in a cumulative manner (0.3, 3, 30 and 300 nmol/L) at 15 minute intervals. To assess the nitric oxide pathway on 17-E2 effects, the strips were similarly precontracted with NE and underwent endothelial NOS blockade by preincubation with L-NAME (10 mmol/L) for 20 minutes. The effective blockade of endothelial NO production was tested in the strips at the absence of acetylcholine-induced relaxation. The involvement of protein synthesis activation by the effect of 17-E2 (genomic mechanisms) was assessed by 20 minutes preincubation with a protein synthesis inhibitor, actinomycin D (10 mmol/L).

To estimate whether 17-E2-induced relaxation is medicated by Maxi-K channel activation, the strip was treated by iberotoxin (10 nmol/L) 10 minutes before the application of 17-E2.

After a concentration response to each agent was recorded, the strip was washed with fresh physiologic solution for 2 or more times over 1 hour and the tension was allowed to relax to baseline level. Results of relaxation studies were expressed as % relaxation of the maximal contraction induced by NE. The concentration response curve of the 17-E2 was constructed by using non-linear curve fitting program (Origin ver 6.0, MicroCal Co., USA).

2.4 Electrophysiological studies

2.4.1 Isolation of single smooth muscle cell

The cavernosal tissue was cut into 1-3 mm pieces in a physiological saline solution (5 mmol/L HEPES, pH 7.4, 140 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2 and 11 mmol/L glucose). To disperse the single cells, the pieces were placed in the Ca2+- and Mg2+-free saline solution containing collagenase (2 mg/mL), trypsin inhibitor (1 mg/mL) and dithioerythritol (5 mmol/L) and digested at 37 for 50 minutes with gentle shaking. Further separation of the cells was achieved by gentle agitation with a wide-bored Pasteur pipette for 3 minutes. Isolated single cells were stored for 30-120 minutes at 4 before each experiment in a K+-rich storage solution consisting of 70 mmol/L glutamic acid, 25 mmol/L KCl, 20 mmol/L taurine, 10 mmol/L oxalic acid, 10 mmol/L KH2PO4, 10 mmol/L HEPES, 0.5 mmol/L EGTA and 11 mmol/L glucose, titrated to pH 7.4 with 1 N KOH.

2.4.2 Patch clamp experiment

Freshly isolated smooth muscle cells were dispersed in a 300 mL chamber on an inverted microscope (IMT-2, Olympus, Japan) and left for 20 minutes. After superfusing for 5 minutes with the physiological saline solution, a relaxed cell with well-contrasted margin was chosen for patch clamp experiments. Patch electrode had 5-20 MW tip resistance when filled with the intrapipette solution containing 140 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L MgATP, 2 mmol/L EGTA, 1 mmol/L CaCl2 and 10 mmol/L HEPES (pH 7.4). For inside-out patches, the intracellular side of the cell membrane was bathed in an external superfusing solution, which was the same as the intrapipette solution except that the Ca2+ concentration was adjusted to 0.1 mmol/L by adding calculated amount of CaCl2. Membrane patches were held at 40 mV in the depolarizing direction for single channel recordings. A patch electrode was attached to the cell with oil-driven micromanipulator (Narishige, Japan) and negative pressure was applied to the electrode. After gigaseal (>5 GW) formation. The inside-out mode of patch clamp technique was established. A standard amplifier (Model AXOPATCH 1-D, Axon, USA) was used and data were fed to a computer (IBM) through a four-pole Bessel type filter at 1 KHz.

2.5 Data analysis

The experiments were repeated more than 5 times at each occasion. Data were expressed as meanSEM. Student's unpaired t-test and one way ANOVA were used to compare the significance of differences and P<0.05 was considered significant. Recorded data from patch clamp experiments were analyzed with pClamp soft ware (Version 6.0, Axon).

3 Results

3.1 Effect of 17-E2 on NE-induced contraction

17-E2 induced relaxation response occurred slowly on 10.32.5 minutes (Figure 1) Cumulative concentrations of 17-E2 elicited concentration-dependent relaxation of the cavernous strips precontracted by NE. The extent of relaxation by 17-E2 of 0.3, 3, 30 and 300 nmol/L was 6.1 %5.1 %, 12.8 %6.5 %, 21.9 %7.6 %, and 39.2 %10.1 %, respectively (Figure 2).

Figure 1. A representative tracing of isometric tension developed in a cavernous strip. Magnitude and timing of response expressed by tension and time bars. Cumulative concentration of 17-estradiol elicits concentration-dependent relaxation of the strip precontracted by norepinephrine.

Figure 2. Relaxation responses to 17b-estradiol in cavernous strips (n=8). Dose-dependent relaxation is noted.

3.2 Effects of L-NAME and actinomycin D on 17-E2 induced relaxation

L-NAME and actinomycin D did not significantly modify 17-E2-induced relaxation (Figure 3 and Figure 4).

Figure 3. Relaxation responses of cavernous strips (n=8) to 17b-estradiol in absence and presence of actinomycin D. There was no difference in relaxation responses between two groups.
Figure 4. 17b-estradiol-induced relaxation responses before and after treatment of L-NAME in cavernous strips (n=8). L-NAME dose not inhibit 17b-estradiol-induced relaxation.

3.3 Effects of iberiotoxin on 17-E2 induced relaxation

Iberiotoxin (10 nmol/L) pretreatment almost completely blocked 17-E2-induced relaxation (Figure 5).

Figure 5. Iberiotoxin (10 nmol/L), the Maxi-K channel blocker, almost completely blocks 17b-estradiol-induced relaxation of cavernous strips (n=8).

3.4 Patch-clamp study

When 3 mmol/L 17-E2 was applied to the intracellular side, the Maxi-K channel activity was increased by about 3-fold (NPo: from 0.810.045 to 2.430.34, n= 5) (Figure 6).

Figure 6. Effect of 17b-estradiol on the Maxi-K channel activity. The patch was held at 10 mV and the free Ca2+ concentration 0.1 mmol/L.
A: Effect of 17b-estradiol observed by adding 3 mol/L 17b-estradiol to intraceullar side of patch.
B: NPo values from 5 separate patches at each condition are shown. bP<0.05, compared with control.

4 Discussion

RT-PCR studies have identified mRNA for both ER and ER in human vascular smooth muscles from the coronary artery, iliac artery, aorta and saphenous vein, but expression of ER tends to predominate in the females [10]. The ER and ER were also expressed in corpora cavernosa in adult rats [8].

E2 acts through binding to its specific receptors localized at cytosolic and nuclear level. Upon binding to E2, ER releases the bound heat shock proteins that maintain the receptor in inactive state, then the ER-ligand complex interacts through DNA binding domain with specific DNA sequence by activating or repressing transcription of target genes [11]. Rapid non-genomic effects have also been demonstrated to be initiated at the plasma membrane level but the nature and characteristics of the mediating receptor is still a matter of debate [12]. In our results, the relaxation response was initiated and maximized within 10 minutes by the application of pharmacological concentration of 17-E2 and the relaxation was not blocked by actinomycin D, suggesting that the relaxation response may be mediated by non-genomic mecha-nism.

Estrogen modulates vascular tone by targeting endothelial cells and/or vascular smooth muscle cells [13]. Estrogen was shown to modulate the synthesis/release of both vasodilators (especially NO) and vasoconstrictors [14]. Estrogen-induced vasodilation is usually presumed to be primarily indirect, i.e., involving release of vasodilatory substances from the endothelium. However, because of the lipid permeability of steroid hormones, it is likely that vascular smooth muscle cells are also physiological targets of estrogen. Estrogen treatment does enhance endothelium-dependent relaxation in femoral, coronary and cerebral arteries [3-5], but other studies have also demonstrated endothelium-independent vasodilatatory responses to estrogen both in vivo [6] and in vitro [7]. These latter studies indicate a direct action of estrogen on the vascular smooth muscles. However, it has been assumed that vasodilatation induced by physiological estrogen concentrations was mediated solely via endothelial cells while only pharmacological concentrations of estrogen stimulated vascular smooth muscle cells directly. In contrast, it is now clear that normal "physiological" concentrations of estrogen (100 - 1000 pmol/L) exert specific endothelium-independent effects on coronary smooth muscle [15-17]. In this study, L-NAME pretreatment did not block the 17-E2- induced relaxation response of rabbit cavernous smooth muscle, suggesting that the relaxation response is NO-independent.

Estrogen attenuates calcium influx [18] and/or stimulates calcium efflux [19] in vascular smooth muscle. Whole-cell patch-clamp studies have demonstrated that estrogen inhibits calcium currents in arterial smooth muscle cells [20]. Estrogen hyperpolarizes vascular smooth muscle by enhancing the potassium conductance. Coronary arteries and isolated coronary myocytes are all consistent with the identification of the Maxi-K channel as an important mediator of estrogen-induced coronary vascular relaxation. Physiological concentrations of estrogen open Maxi-K channels in cultured human coronary artery myocytes in the absence of endothelium [17]. In contrast to single myocytes from ovine, porcine or human arteries, Maxi-K channels of rat coronary myocytes do not respond directly to estrogen [4].

Micromolar estrogen can open Maxi-K channels in an artificial expression system in the absence of endothe-lium, nitric oxide (NO) or cGMP [21], but whether this direct effect of estrogen on Maxi-K channel proteins is important in vivo remains to be established. In contrast, effects of estrogen on Maxi-K channels in porcine coronary arteries were mediated by cGMP stimulation of the cGMP-dependent protein kinase [7]. In our inside-out patch clamp study, 17-E2 activated Maxi-K channel and iberotoxin pretreatment completely blocked the 17-E2-induced relaxation, suggesting that the relaxation response of rabbit cavernous smooth muscle is mediated by the Maxi-K channel.

In conclusion, 17-E2 may be involved in relaxation of the rabbit cavernous smooth muscles via a non-genomic and NO-independent mechanism. 17-E2 stimulates the Maxi-K channel of rabbit cavernous myocyte directly.

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Correspondence to: Sae-Chul Kim, M.D, Ph.D. Department of Urology, Chung-Ang University Yongsan Hospital, 65-207 Hangang-Ro 3-Ka, Yongsan-Ku, Seoul 140-757, Korea.
Tel/Fax: +82-2-792 8496
E-mail: saeckim@unitel.co.kr
Received 2003-06-05 Accepted 2004-04-21