Introduction

Penile erection is a complex neurovascular event involving the interaction of the central nervous system (CNS), the peripheral nervous system and penile arterial and trabecular smooth muscles.^{1, 2} Penile tumescence and detumescence are hemodynamic events that are controlled by the nervous system. The CNS integrates and coordinates incoming sensory information from the visual and auditory system and congestive/imaginative stimuli, along with tactile and olfactory information.^{1, 2}

Historically, some dopamine receptor agonists were suggested to alter sexual behavior.^{3, 4} Apomorphine, a nonselective dopaminergic receptor agonist used in the treatment of erectile dysfunction (ED), has been shown to be effective in eliciting penile erection in man and animal models^{5, 6} and its clinical utility as an erectogenic agent has been studied for more than a decade. Early pharmacological studies indicated that two major families of G-protein-coupled dopamine receptors existed, and activation of the receptors either stimulated (D1-like receptor) or inhibited (D2-like receptor) synthesis of cAMP.⁷ The use of the D1/D2 dopamine receptor agonists for the treatment of ED provides strong support in favor of a participation of the dopaminergic system in the control of sexual function. The mechanism of erectile responses to dopamine receptor agonists is postulated to centrally stimulate dopamine receptors in paraventricular nucleus and medial preoptic area of the hypothalamus.^{8, 9, 10}

Besides known mechanisms of central-acting effect of dopamine agonists, recent studies showed that human corporal smooth muscle cells possess both D1- and D2-like receptors and that dopamine receptor agonists have direct peripheral relaxant effects on human corpus cavernosum.¹¹ However, the effect of peripheral dopamine and signal transduction pathways in modulating the vascular tone of the penis has not been completely elucidated.

Penile erection is a hemodynamic event, which is coordinated with corporal smooth muscle relaxation. Decreased penile vascular resistance induced by corporal smooth muscle relaxation is the most important step in penile erection. The modulation of corporal smooth muscle tone is a complex process requiring the integration of a host of intracellular events and extracellular signals. The potassium channels have been shown to play an important role in both the physiological and pathological regulation of smooth muscle tone in diverse tissues¹² and recent studies both in vitro and in vivo have documented the importance of potassium channels to the modulation of corporal smooth muscle tone.^{13, 14, 15, 16}

We investigated the mechanism of action of peripheral dopamine receptor agonists in regulating the tone of the corpus cavernosum, and we studied the effect of dopamine receptor agonists on potassium channel and their signal transduction pathway in corporal smooth muscle cells.

Materials and methods

Explant cell cultures

All studies were performed according to a protocol approved by the Internal Review Board of Samsung Medical Center, Sungkyunkwan University School of Medicine. Human erectile tissue was obtained from the corpus cavernosum of patients undergoing surgery for implantation of penile prostheses. Homogeneous explant cell cultures of human corporal smooth muscle cells were prepared as previously described.^{17, 18, 19} Briefly, radial sections approximately 3 3 10 mm were excised from the mid-penile shaft of each patient; these specimens consisted exclusively of smooth muscle, endothelium and connective tissue, with occasional nerve fibers. Tissue was washed, cut into 1- to 2-mm pieces, and placed in tissue culture dishes with a minimal volume of Dulbecco's medium (Gibco Invitrogen, Carlsbad, CA, USA) with 20% fetal calf serum. After the tissue was allowed to attach to the plate (usually 1–2 days), additional medium was added. Smooth muscle cells migrated from the explant and underwent division. Cells were subsequently detached using a Trypsin/ethylenediaminetetraacetic acid protocol to establish secondary cultures from the explants. These cultures were morphologically homogeneous, and furthermore, we did not observe cobblestone morphologies characteristic of endothelial cells or the very flattened and spread out shapes characteristic of fibroblasts. Cellular homogeneity was further verified by the presence of smooth muscle-specific -actin immunoreactivity. Cultures were maintained for no more than four passages; importantly, during this time all measured pharmacological and molecular properties observed in the intact tissue were retained in culture.

Electrophysiological recordings

Single-channel recordings using the cell-attached configuration and whole-cell patch-clamp technique were performed. The patch electrodes were made from borosilicate glass capillary tubing (World Precision Instruments, Sarasota, FL, USA) and had resistances of 3–5 M. The cell suspension was placed into a small chamber (0.6 ml) on the stage of an inverted microscope (TMD Diaphot, Nikon, Tokyo, Japan). Experiments were performed using a patch-clamp amplifier (Axopatch-lD, Axon Instruments, Foster City, CA, USA). Current signals were filtered at 1 kHz and sampled at 5 kHz, digitized by an AD converter (TL-1, Axon Instruments) and analyzed on a personal computer using the pCLAMP software (version 6.0.2, Axon Instruments). The voltage-clamp experiments were performed by applying both ramp pulses and step pulses. The liquid junctional potential between the pipette solution and Tyrode solution was only about 3 mV, and it was not corrected. Series resistance (about 6–10 M) and capacitative currents also were not compensated, because the cell size and measured currents were relatively small. In each experiment, whole-cell configuration was not made until the seal resistance became larger than 5 G. The membrane capacitance was determined from the current amplitude elicited in response to hyperpolarizing voltage ramp pulses from a holding potential (HP) of 0 to -5 mV (duration 25 ms at 0.2 V s^-1); this procedure avoided interference by any time-dependent ionic currents. Average cell capacitance was 35.32.6 pF (n=44).

Drugs and solutions

For the whole-cell experiments, the bath (external) solution contained (in mM): 135 NaCl, 5 KCl, 1.2 MgCl₂, 1.2 CaC1₂, 10 glucose, 10 HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonicacid)), and the pH was adjusted to 7.4 with NaOH. The pipette (internal) solution contained (in mM): 140 KCl, 10 HEPES, 2 K₂ATP, 3.3 CaCl₂, 2 MgCl₂, 5 EGTA (ethyleneglycol-bis-(2-aminoethyl ether)-N,N'-tetraacetic acid), 1 GTP, and the pH was adjusted to 7.2 with KOH. For single-channel recordings (cell-attached configuration), the composition of both pipette and bath solution was the same (in mM): 140 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, 10 HEPES and the pH was adjusted to 7.3 with KOH. All chemicals were obtained from Sigma Chemical Company (St Louis, MO, USA).

Measurement of cyclic nucleotide levels

Intracellular cyclic AMP (cAMP) was determined by a commercially available enzyme immunoassay (EIA) kit (Biotrak cAMP enzyme immunoassay RPN 225; Amersham Biosciences, Little Chalfont, Bucks, UK). For assay, corporal smooth muscle cells were washed three times with phosphate-buffered saline, and then fresh serum-free medium was added. The cells were pretreated for 30 min with 0.1 mM isobutylmethylxanthine (IBMX), nonselective phosphodiesterase (PDE) inhibitor and followed by addition of various concentrations of dopamine (10, 50 and 100 M). cAMP levels were also measured in control and the cells were stimulated by increasing concentrations of apomorphine, dopamine receptor agonist, by EIA. For the cyclic GMP (cGMP) experiments, the cells were stimulated by addition of agonists in the presence of 30 M zaprinast. After 30 min of incubation, the medium was quickly aspirated, and the reactions were stopped by adding ice-cold 65% ethanol. The extract was evaporated and intracellular cGMP levels were measured by iodinated radioimmunoassay kit (cGMP RIA kit RPA 525; Amersham Biosciences, Little Chalfont, Bucks, UK).

Data analysis

The data are expressed as means.e.m. Statistical analyses were carried out by Student's t-test when applicable. Significance was established at P<0.05. There were at least four experiments for each data point.

Results

Effects of dopamine receptor agonists on the BK_Ca channels in the whole-cell configuration

The whole-cell K⁺ currents were recorded in cultured human corporal smooth muscle cells. At the -60 mV HP, dopamine (10 M), nonselective dopaminergic receptor agonists (D1 and D2), was added to the bath solution. Ramp currents were induced by the three ramp potential pulses from -60 to 60 mV for 400 ms. As illustrated (Figure 1), dopamine significantly increased whole-cell K⁺ currents by 283.555.7% (at +60 mV; n=12, P<0.001). Dopamine increased K⁺ currents at ramp potential pulses and K⁺ currents were completely inhibited by tetraethylammonium (TEA; 1 mM) or charybdotoxin (100 nM). From these results, it was confirmed that whole-cell currents were through BK_Ca channels.

Figure 1.

Current–voltage relationships obtained using a 400 ms ramp pulse from -60 to 60 mV. (a) Time course of 10 M dopamine-activated K⁺ currents. (b and c) Dopamine-stimulated currents were inhibited by 1 mM tetraethylammonium (TEA) or 100 nM charybdotoxin, BK_Ca channel-selective inhibitors.

Full figure and legend (33K)

Extracellular application of apomorphine significantly increased whole-cell K⁺ currents in a concentration-dependent fashion. And whole-cell K⁺ currents were increased by 292.458.8% (at +60 mV; n=9, P<0.005) after application of 10 M apomorphine (Figure 2). We also confirmed that the increase in whole-cell currents was mainly due to activation of the TEA-sensitive BK_Ca channels. There was no statistically significant difference in activated K⁺ currents between dopamine and apomorphine at the same concentration (10 M).

Figure 2.

Current–voltage relationship; currents were evoked by ramp pulse from -60 to 60 mV (400 ms). (a) Membrane currents were recorded before (control) and 10 min after application of 10 M apomorphine. (b) Apomorphine-stimulated outward currents were almost completely inhibited by 1 mM tetraethylammonium (TEA). (c) Summary of increasing currents measured in reconstitution experiments under different experimental conditions. Data are mean of percent increases.e.

Full figure and legend (83K)

Single-channel characteristics of BK_Ca channels in the presence of dopamine receptor agonists

The unitary potassium channel currents were observed in cell-attached patches of human corporal smooth muscles, and HP was 60 mV. The BK_Ca channel currents were recorded from the same cells before and 10 min after the application of 10 M dopamine. The dopamine activated BK_Ca currents in cultured human corporal smooth muscle cells. As illustrated in Figure 3A(a), the channel openings in cell-attached mode were significantly increased by dopamine (10 M). To determine whether the stimulatory effect of dopamine on BK_Ca channel activity was mediated though -adrenoceptors, cells were pretreated for 30 min with propranolol (10 M). Inhibition of -adrenoceptors did not influence the stimulatory effects of dopamine on BK_Ca channels in cell-attached patches (Figure 3A(b)). Treating cells with apomorphine showed the same effects of dopamine on BK_Ca channel activity (Figure 3B). Apomorphine increased BK_Ca channel activity in a concentration-dependent fashion.

Figure 3.

Experiment was carried out in a cell-attached patch at a holding potential (HP) of 60 mV. (A) (a) Recording from the same cell before and 10 min after application of 10 M dopamine. (b) In the presence (30 min preincubation) of 5 M propranolol, subsequent 10 min exposure to 10 M dopamine. (B) Effect of various concentration of apomorphine on channel activity. c, Channel close level.

Full figure and legend (121K)

Effects of dopamine receptor agonists on intracellular cyclic nucleotide levels

In the absence of PDE inhibitors, treatment of cells with dopamine for 30 min resulted in increased intracellular cAMP concentrations in a concentration-dependent fashion, and pretreatment of 0.5 M of propranolol did not disturb the effects of dopamine (Figure 4A(a)). As indicated in Figure 4A, cells were treated with 100 M IBMX, nonselective PDE inhibitor, for 30 min prior to the addition of dopamine to inhibit cAMP hydrolysis; and greater response of cAMP could be induced by dopamine. Treatment of cells with apomorphine for 30 min also resulted in increased intracellular cAMP concentrations in a concentration-dependent fashion (Figure 4(b)). Forskolin, a direct adenylyl cyclase activator, resulted in marked increase in intracellular cAMP concentrations and used as a positive control. In contrast to their effects on cAMP accumulation, dopamine did not increase the cGMP levels (Figure 4B).

Figure 4.

(A) (a, left) Cells were exposed to dopamine (10, 50 and 100 M) for 30 min in the presence (30 min preincubation) with 0.5 M propranolol or without. (a, right) In the presence (30 min preincubation) of 0.1 M isobutylmethylxanthine (IBMX), subsequent exposure to dopamine for an additional 30 min. (b) Cyclic AMP (cAMP) levels before (control) and 30 min after exposure to either increase concentrations of apomorphine or forskolin. (B) Cyclic GMP (cGMP) levels before (control) and 30 min after exposure to either 100 M dopamine or 10 M single nucleotide polymorphism (SNP). Data are mean cAMP/cGMP concentration of four separate experiments s.e.

Full figure and legend (106K)

Discussion

This study demonstrated that dopamine receptor agonists activated BK_Ca channels in corporal smooth muscle cells and this response was mediated by the accumulation of cAMP. These results implied that besides known mechanisms of central-acting effect of dopamine agonists, the activation of BK_Ca channels in corporal smooth muscle cells might be one of mechanisms in inducing penile erection.

Recent studies indicate that dopamine modulates the opening of large conductance, calcium- and voltage-activated potassium channels (BK_Ca channels) in bovine adrenal medullary chromaffin cells²⁰ and porcine coronary arterial myocytes.²¹ Han et al.²¹ showed that activation of dopamine receptors causes stimulation of BK_Ca channel activity. They also demonstrated that dopamine-induced coronary vasodilation was mediated by cAMP-dependent stimulation of protein kinase G (PKG). Like other vascular smooth muscle cells, smooth muscle cells from human corpus cavernosum possess substantial outward K⁺ currents primarily composed of K⁺ efflux through BK_Ca channels. Because of their large conductance and high density of expression, these channels help set and maintain the resting membrane potential of human corpus cavernosal smooth muscle.²² Moreover, TEA (1 mM) that is a selective blocker of large-conductance calcium-activated potassium channels (BK_Ca channels) at this concentration and charybdotoxin (100 nM) that inhibits both intermediate and large-conductance calcium-activated potassium channels at the concentration induces contraction of isolated corpus cavernosal smooth muscle, thus underscoring the importance of BK_Ca channels in regulating corporal smooth muscle tone even under nonstimulated conditions.²³ When intracellular Ca²⁺ levels increase during contraction, BK_Ca channels provide an important repolarizing negative-feedback mechanism that helps reverse active contraction. In our study, we confirmed that extracellular application of dopamine and apomorphine significantly increased the whole-cell currents and single-channel currents mainly due to activation of the TEA-sensitive BK_Ca channels. Together with previous report that apomorphine could act peripherally as relaxing agent on human corpus cavernosal strips in an endothelium-independent manner,²⁴ the activation of BK_Ca channels by dopamine receptor agonists can be one of the mechanisms by which dopaminergic agent has peripheral proerectile effect.

Multiple subtypes of G-protein-coupled dopamine receptors are found in a variety of peripheral organs and the CNS, and dopamine is a nonselective catecholamine that can stimulate both classes of dopamine receptors (D1 and D2) as well as - and -adrenoceptors.^{25, 26} Vascular smooth muscle expresses both dopaminergic and adrenergic receptors; thus it was possible that dopamine-induced relaxation of corporal smooth muscles might be mediated via dopamine receptors and/or -adrenoceptors, which also open BK_Ca channels in vascular^{27, 28} and nonvascular smooth muscle cells.^{29, 30} In our study, propranolol, -adrenoceptor inhibitor, did not diminish the stimulatory action of 10 M dopamine on BK_Ca channels in corporal smooth muscle cells. Thus the effect of dopamine (10 M) cannot be attributed to activation of -adrenoceptors; however, the possibility that higher concentrations of dopamine might activate these or other receptors cannot be excluded. These findings suggest that the effects of dopamine on BK_Ca channel activity are mediated through dopamine receptors, and that this transduction mechanism underlies corporal smooth muscle relaxation to dopamine receptor agonists.

Our findings indicate that dopamine-induced relaxation of human corpus cavernosum seems to involve dopamine receptors and BK_Ca channels because (1) application of dopamine receptor agonists increased BK_Ca channel activity and this effect was not disturbed by propranolol, -adrenoceptor antagonist and (2) blockade of BK_Ca channels inhibits dopamine-stimulated BK_Ca channel activity.

Although we had obtained convincing evidence that linked activation of dopamine receptors with stimulation of BK_Ca channels in corporal smooth muscles, it remains unclear by which cascade this channel activity was mediated. Our study showed that treatment of cells with dopamine receptor agonists resulted in increased intracellular cAMP concentrations in a concentration-dependent fashion. From these results, it is inferred that the stimulatory action of dopamine agonists to BK_Ca channels is via a cAMP-dependent mechanism. To clarify the downstream events, however, leading to opening of BK_Ca channels and subsequently to relaxation of corporal smooth muscles, the further investigation to find out the role of cAMP and protein kinases will be necessary.

Conclusions

We first showed that dopamine receptor agonists activated opening of BK_Ca channels in corporal smooth muscle cells, and this response was mediated by the accumulation of cAMP. Besides known mechanisms of central-acting effect of dopamine agonists, the activation of BK_Ca channels in corporal smooth muscle cells might be one of the mechanisms in inducing penile erection.

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Acknowledgements

We thank Soo Jung Lee, a student of Brooks School, for her assistance with translation.