¹Department of Urology, University Medical Centre of Cologne, Cologne, Germany

²Institute I of Anatomy, University Medical Centre of Cologne, Cologne, Germany

Correspondence to: F Sommer, Klinik und Poliklinik für Urologie der Universität zu Köln, Joseph-Stelzmann-Str. 9, D-50924 Köln, Germany. E-mail: Frank.Sommer@uni-koeln.de

Nitric oxide (NO) is an important mediator in the cavernosal smooth muscle relaxation that causes erections. The purpose of this study was to examine the existence, distribution and phosphorylation stage of two recently discovered key enzymes for NO regulation in human cavernosal tissue, the MAP Kinase 1/2 (Erk 1/2) and the serine/threonine specific protein kinase Akt/PKB. The expression of the enzymes was examined in corpus cavernosum specimens taken from both potent men and from patients with long-term impotence. There was a distinct difference in the activation stage of the MAP Kinase 1/2 (Erk 1/2) between endothelium and smooth muscle cells in potent patients. This finding gives evidence for a cell-type-specific regulation of the eNOS-dependent NO release. Furthermore, we found a higher basal level of active MAP Kinase 1/2 (Erk 1/2) in impotent patients. This finding gives the first evidence for an inhibitory influence of MAP Kinase 1/2 (Erk 1/2) on cavernosal eNOS activity.

International Journal of Impotence Research (2002) 14, 217-225. doi:10.1038/sj.ijir.3900856

corpus cavernosum; MAP Kinase 1/2 (Erk 1/2); serine/threonine specific protein kinase Akt/PKB

Introduction

Penile erection is a hemodynamic process, involving increased arterial inflow and restricted venous outflow, in coordination with corpus cavernosum smooth muscle relaxation.¹ Although this process is generally accepted to be under neuroregulatory control,² biochemical mediators released locally from the endothelium and/or smooth muscle also participate in initiating and maintaining erection.³ Nitric oxide (NO), which is produced both in cavernosal nerves and the endothelium, has been recognized as playing a key role in the physiology of penile erection.² Additionally, it has been shown that endothelial NO synthase (eNOS) is one of the main sources of NO in the cavernosal tissue, which is then available in endothelial cells and cavernosal smooth muscle cells.⁴ Nitrinergic innervation and eNOS expression have shown a broad heterogeneity; up until now, no correlation between eNOS expression and erectile function has been observed.⁵ Recently it was shown that the NO release through eNOS is modulated by phosphorylation of the enzyme, mediated by MAP Kinase 1/2 (Erk 1/2) and serine/threonine specific protein kinase Akt/PKB.^6,7 It has been demonstrated that the serine/threonine specific protein kinase Akt/PKB enzyme plays an important role in the activation of eNOS. Mimicking the phosphorylation of Ser 1177 directly enhances enzyme activity and alters the sensitivity of the enzyme to Ca²⁺, rendering maximum enzyme activity at sub-physiological concentrations of Ca²⁺. Thus, phosphorylation of eNOS by serine/threonine specific protein kinase Akt/PKB represents a novel Ca²⁺-independent regulatory mechanism for the activation of eNOS. Constitutively active serine/threonine specific protein kinase Akt/PKB stimulates the phosphorylation of eNOS, but the inactive kinase does not. As only a few substrates for the serine/threonine specific protein kinase Akt/PKB have been described so far, the results reported identify eNOS as a novel Akt target.⁶ While active Akt/PKB increases the eNOS-mediated NO release, the activation of MAP Kinase 1/2 (Erk 1/2) leads to a reduction of eNOS activity through an as yet unknown phosphorylation process. A direct interaction between these kinases seems unlikely because the inactivation of the putative Akt/PKB phosphorylation state does not block eNOS phosphorylation by MAP Kinase 1/2 (Erk 1/2).⁷ NO production in endothelial cells can be a response to different stimuli, such as acetylcholine,⁸ shear stress, bradykinin, substance P and adenosine diphosphate (ADP).⁹ For acetylcholine, stimulation of NO release derived from eNOS not located in the endothelium was also found up to now.¹⁰

Acetylcholine, one of the key transmitters for the erectile function,¹¹ has been shown to act as a relaxant stimulus in the corpora cavernosa of rats, by means of activation of nNOS and eNOS-derived NO release.¹² Other researchers were able to demonstrate acetylcholine as an activator of Akt/PKB and MAP Kinase 1/2 (Erk 1/2).^13,14 Thus, we considered Akt/PKB and MAP Kinase 1/2 (Erk 1/2) as possible upstream regulators of eNOS in the corpus cavernosum, depending on the expression and distribution of these enzymes.

Up to now, cavernosal tissue has not been examined for the MAP Kinase 1/2 (Erk 1/2) and the serine/threonine specific protein kinase Akt/PKB enzymes. The aim of this study was to investigate the distribution and basal activation stage of the MAP Kinase 1/2 (Erk 1/2) and the serine/threonine specific protein kinase Akt/PKB in the human corpus cavernosum. In this context, we examined cavernosal tissues from both potent and impotent patients, in order to elucidate possible differences between these groups.

Patients and methods

Patients and collection of tissue

Twenty-four human corpus cavernosum tissue specimens were obtained from patients subjected to penile surgery, with the patients' informed consent. The specimens were immediately fixed in 4% paraformaldehyde for 4 h and then rinsed in 0.1 M phosphate-buffered saline (PBS) for 24 h. The tissue was stored for 12 consecutive hours in a PBS solution containing 18% sucrose for cryoprotection and then frozen to -80°C.

Fourteen of the patients (age range 16-63 y; mean age 42 y) with normal erectile function suffered from penile deviations. All patients were treated via Nesbit's surgical procedure. Normal erectile function was ascertained by anamnestic evaluation. Seven patients (age range 27-66 y; mean age 46 y) had shown complete erectile dysfunction for more than 2 y. This group underwent implantation of flexible hydraulic penile prostheses. Four patients suffered from severe venous leakage, and one patient from impotence due to radical surgery of a bladder carcinoma. Two patients had diabetes and a combined arterial-venous insufficiency of the corpus cavernosum. Three patients underwent transsexual operations (male-female). These patients had undergone a long-term hormonal pre-treatment, but reported normal erections (Table 1).

Immunohistochemistry

Prior to immunohistochemical examination, tissue sections (6 µm) were placed in a bathing solution of 3% H₂O₂ and methanol for 20 min, then permeabilized for 10 min with 0.25% Triton-X in 0.1 M TBS. The sections were then treated with 5% bovine serum albumin (BSA) solution in TBS. Before each step, the sections were rinsed three times in TBS buffer. Incubation with the primary antibody occurred in a TBS-based solution of 0.8% BSA for 24 h at 4°C. The anti-Erk 1/2 antibody was applied in a dilution of 1:400 and the anti-Akt kinase antibody was applied in a dilution of 1:150. The antibodies for the phosphorylated (activated) forms of the enzymes were applied in a dilution of 1:400 (for activated Map Kinase 1/2 (for Erk 1/2) and 1:500 (activated AKT/PKB). After rinsing with TBS, the sections were incubated with the corresponding secondary biotinylated antibody for 1 h at room temperature. A streptavidin-horseradish peroxidase complex was then applied as a detection system (1:150 dilution) for 1 h. Finally, the staining was developed for 3-10 min with 150 µl 3,3-diaminobenzidine tetrahydrochloride (DAB), 150 µl NH₄Cl, 50 µl glucose oxidase, 300 µl 10% glucose, and 300 µl NiSO₄ in 15 ml phosphate buffer (PB). Negative control sections were incubated without the primary antibody.

Immunoblot

Homogenisation: Human corpus cavernosum was obtained fresh from the operating room and frozen in fluid nitrogen. Tissue was homogenised and treated with dissection buffer pH 7.4 (240 mM sucrose, 1 mM PMSF, 20 mM PIPES, 10 mM EDTA, 50 mM NaH₂PO₄). Protein concentration was determined according to Bradford.¹⁵

SDS-PAGE, immunoblotting and detection: SDS-PAGE was performed according to Laemmli.¹⁶ Protein samples were denatured by boiling in 0.8% SDS and 0.5% -mercaptoethanol (v/v) and electrophoresed in a 12.5% gel. For immunoblotting, a semi-dry system (SemiDryBlotter II, KEMENTEC, Copenhagen, Denmark) was used. Proteins were blotted on a PVDF membrane (0.2 micron, BioRad Laboratories, Munich, Germany) for 1 h at 170 mA. The membrane was blocked with 1% powdered milk in buffer I pH 7.8 (25 mM Tris/HCl, 137 mM NaCl, 3 mM KCl, 0.5% Tween 20) for 1 h at RT (room temperature). The primary antibodies, rabbit anti-phospho-AKT-1/PKB [Thr 308] (1:1000), rabbit anti-AKT-1/PKB (1:400), rabbit anti-MAP kinase 1/2 [ERK 1/2] (1:1200) and mouse anti-MAP Kinase, activated [diphosphorylated ERK 1/2] (1:1000) in buffer I, were incubated over night at 4°C. After washing (3´15 min.) with buffer I, the relevant secondary antibodies were used for 1 h at room temperature (1:800, in buffer I+0.1% powdered milk). Following washing, steps were performed before incubation with streptavidin-conjugated HRP (1:300, Amersham, in buffer I+0.1% powdered milk) for 1 h at RT. After washing (3´15 min.) with buffer II pH 7.8 (buffer I with 0.05% Tween 20), the detection was prepared with DAB solution.

Materials

The primary antibodies, rabbit anti-phospho-AKT-1/PKB [Thr 308] (1:1000, Upstate Biotechnology, Lake Placid, NY, USA), rabbit anti-AKT-1/PKB (1:400, Upstate Biotechnology), rabbit anti-MAP kinase 1/2 [ERK 1/2] (1:1200, Upstate Biotechnology), mouse anti-MAP Kinase, activated [Diphos-phorylated ERK 1/2] (1:1000, Sigma, Saint Louis, MO, USA), goat anti-rabbit conjugated biotin (1:400, Dako, Glostrup, Denmark), goat anti-mouse conjugated biotin (1:400, Dako) and streptavidin-conjugated HRP (1:150, Amersham, Life Science, Bucks, UK) were used.

Results

Immunoblot analysis

The immunoblot analysis reveals that the MAP kinase 1/2 (Erk 1/2) and serine/threonine specific protein kinase Akt/PKB are expressed in human corpus cavernosum. Furthermore, it is recognizable that a part of the expressed kinases were in an activated stage in the tissue from the non-erect corpus cavernosum. Additionally, it was demonstrated that active MAP Kinase 1 (Erk 1) is present in greater quantity than active MAP Kinase 2 (Erk 2). Inactive MAP Kinase 1/2 (Erk 1/2) has a similar expression level (Figure 1).

Immunohistochemistry

To identify the cellular localization of the Akt/PKB and MAP Kinase, as well as their activation stage, in the different cell compartments of the cavernosal tissue, immunohistochemical stainings were performed. In all specimens of cavernosal tissue, a distinct immunoreactivity of inactive and active MAP Kinase 1/2 (Erk 1/2) and both inactive and active serine/threonine specific protein kinase Akt/PKB was observed in different cellular compartments.

We found the inactive and active MAP Kinase 1/2 (Erk 1/2; Figures 3, 4 and 5) and the inactive and active serine/threonine specific protein kinase Akt/PKB (Figures 2, 4, 5) in the endothelium of the cavernosal sinus, in the endothelium of the cavernosal arteries, in the vascular and cavernosal smooth muscle and in cavernosal nerve fibres of patients both without (Figures 2, 3 and 4) and with (Figure 5) erectile dysfunction. The endothelial expression of the MAP kinase 1/2 (Erk 1/2) and the serine/threonine specific protein kinase Akt/PKB was more pronounced than the muscular expression of both kinases (Figures 2A, 3A and 4A, B). Comparable differences in the amount of the active serine/threonine specific protein kinase Akt/PKB were observed between endothelial cells and cavernosal and vascular smooth muscle cells (Figures 2B and 4C). A distinct amount of serine/threonine specific protein kinase Akt/PKB was found in the activated form in smooth muscle cells and endothelial cells (Figures 2B and 4C). While the amount of active MAP kinase 1/2 (Erk 1/2) was also high in the endothelium, in the smooth muscle cells of fibromuscular stroma, only a faint immunoreaction for active MAP kinase 1/2 (Erk 1/2) was seen in potent patients (Figures 3B). Akt/PKB and MAP kinase were also found in cavernosal nerve fibres; here, the immunoreaction for both activated kinases was high (Figure 4E and F).

No differences between potent and impotent patients could be discovered for Akt/PKB (Figures 2A and 5A) and MAP kinase 1/2 (Erk 1/2) (Figures 3A and 5B) in the endothelium and cavernosal and vascular smooth muscle. All specimens from impotent patients with various primary diseases showed a higher amount of the active MAP kinase 1/2 (Erk 1/2) in the smooth muscle of cavernosal fibromuscular stroma than in potent patients (Figure 5D), while no differences could be observed for the active Akt/PKB (Figure 5C).

Discussion

Penile erection is produced by an increased blood flow to the corpus cavernosum (CC), made possible by the opening of penile resistance vessels (helicine arteries), the relaxation of the CC cells, and occlusion of the venous outflow.¹ The erectile response in several animal models depends on NO, produced by nerves as well as by the vascular endothelium.^1,17,18 NO activates soluble guanylate cyclase, which leads to the production of cyclic GMP (cGMP). cGMP signals via three different receptors in eukaryotic cells, including ion channels, phosphodiesterases, and protein kinases.¹⁹

There is convincing evidence that during erection the local release of NO and/or related factors produces relaxation of the human corpus cavernosum.² Non-adrenergic, non-cholinergic (NANC) nerve-mediated NO release appears to be the most important factor with respect to cavernosal smooth muscle relaxation.¹⁷ NO has the capacity to activate guanylate cyclase. Submicromolar concentrations of NO cause rapid and robust increases in cGMP levels in target cells.²⁰ Changes in the cellular cGMP level as a secondary messenger lead to a physiological response in these target cells.

Smooth muscle relaxation is one of the most important effects of the NO/cGMP pathway with regard to erectile function.⁵ Recently it has been shown that endothelial NO synthase (eNOS) is one of the most important sources of NO in cavernosal smooth muscle cells.^4,21 Furthermore, it has been demonstrated that erectile dysfunction cannot be reduced solely to pathological findings in penile NO synthases, since no correlation was found between the expression of the NO synthases and erectile function in the cavernosal tissue of potent and impotent patients.⁴ However, a functional impairment of eNOS activity may lead to impairment of cavernosal smooth muscle relaxation in response to endothelium-mediated stimuli in hypercholesterolemic rabbits.²² Thus it could be speculated that alteration of eNOS regulation leads to the impairment of NO-dependent erectile function.

For an understanding of the complete signal transduction in the corpus cavernosum, it is necessary to elucidate the whole NO/cGMP pathway with its potential upstream and downstream target proteins. In recent studies we dealt with the expression of the NOS isoforms and the molecules involved in the downstream pathway of NO in human cavernosal tissue from potent and impotent patients.^4,5 In this study we focus on recently recognized upstream regulators of eNOS. To our knowledge, this is the first study that deals with the expression of MAP kinase 1/2 (Erk 1/2) and serine/threonine specific protein kinase Akt/PKB and their basal activation stage in the human corpus cavernosum.

We have found a high expression of MAP Kinase 1/2 (Erk 1/2) and serine/threonine specific protein kinase Akt/PKB, as well as a distinct amount of the activated form of these kinases, in the endothelium of the cavernosal sinus, in the endothelium of the cavernosal vessels and in cavernosal nerve fibres. These kinases are directly involved in eNOS regulation. Therefore, it can be postulated that the activation of eNOS leads to increased NO production, which results in smooth muscle relaxation with consequent erection.

The activation mechanism for eNOS is not yet completely understood. It was demonstrated in human endothelial cells that the serine/threonine protein kinase Akt/PKB^23,24,25 mediates the activation of eNOS, leading to increased NO production. Inhibition of the phosphatidylinositol-3-OH kinase/Akt pathway or mutation of the Akt side of the eNOS protein (at serine 1177) attenuates the serine phosphorylation and prevents the activation of eNOS. Mimicking the phosphorylation of Ser 1177 directly enhances enzyme activity and alters the sensitivity of the enzyme to Ca²⁺, rendering maximum enzyme activity at sub-physiological concentrations of Ca²⁺. Thus, phosphorylation of eNOS by Akt/PKB represents a novel Ca²⁺-independent regulatory mechanism for activation of eNOS.⁶

It has been demonstrated that over-expression of the constitutively active Akt/PKB results in a 1.4-fold increase in intracellular cGMP, indicating that eNOS can be activated by an Akt/PKB-dependent pathway.⁶

The involvement of serine/threonine specific protein kinase Akt/PKB in eNOS phosphorylation has been effectively established in several independent studies.^6,26,27 Constitutively active serine/threonine specific protein kinase Akt/PKB stimulates the phosphorylation of eNOS, but the inactive kinase does not. As only a few substrates for the serine/threonine specific protein kinase Akt/PKB have been described so far, the results reported identify eNOS as a novel Akt target.⁶ Our study showed endothelial and smooth muscle expression of the serine/threonine specific protein kinase Akt/PKB. Active serine/threonine specific protein kinase Akt/PKB was found in endothelium and cavernosal and vascular smooth muscle cells. The sinus endothelium and the cavernosal smooth muscle are the source of eNOS-mediated NO release in the corpus cavernosum.⁵ So it can be speculated that the activation of eNOS by serine/threonine specific protein kinase Akt/PKB in the corpus cavernosum leads to increased NO production, resulting in smooth muscle relaxation with consequent erection (Figure 6). The finding of a distinct amount of active serine/threonine specific protein kinase Akt/PKB in cavernosal smooth muscle cells and the sinus endothelium hints at a possibly major role played by the NO pathway in achieving erection.

eNOS is reversibly associated with protein kinases that are implicated in opposing regulatory effects on the enzyme, leading to both eNOS activation (Akt) and inhibition (Erk).⁷

There is evidence for the direct involvement of MAP Kinase 1/2 (Erk 1/2) in eNOS regulation. Showing that eNOS can be phosphorylated (activated) by MAP kinase 1/2 (Erk 1/2) seems highly likely, since several additional residues in eNOS undergo phosphorylation in endothelial cells. This suggests that diverse kinase pathways might have an important influence on the enzyme. Studies by Bernier et al. provided evidence indicating that MAP kinase 1/2 (Erk 1/2) plays a key role in eNOS regulation.⁷ It was strongly suggested that phosphorylation of eNOS catalysed by MAP kinase 1/2 (Erk 1/2) can lead to enzyme inhibition, and it was shown that in vitro phosphorylation of eNOS by MAP kinase 1/2 (Erk 1/2) is associated with a reduction in enzyme activity. MAP kinase 1/2 (Erk 1/2) inhibits eNOS by phosphorylating the enzyme in endothelial cells.⁷ Our study showed endothelial and smooth muscle expression of the MAP kinase 1/2 (Erk 1/2). This result makes it likely that MAP kinase 1/2 (Erk 1/2) plays a role as negative regulator of eNOS activity in the cavernosal tissue (Figure 6).

In our study, we did not find any distinct differences between potent and impotent patients with regard to the expression and distribution of MAP kinase 1/2 (Erk 1/2). However, the specimens from impotent patients with various primary diseases showed a higher expression of the active MAP kinase 1/2 (Erk 1/2) in the smooth muscle of cavernosal fibromuscular stroma under the observed basal conditions than in potent patients. It can be speculated that the higher activation stage of MAP kinase 1/2 (Erk 1/2) in cavernosal smooth muscle of impotent patients impairs the switch-on of eNOS by erectile stimulation. This could identify a possible pathway affecting erectile dysfunction.

We have to stress the limitations of our study with respect to the small number of patients, which did not allow for a statistical analysis. Additionally, it should be stressed that our study shows the expression or distribution of the kinases, but only the basal functional status. Further studies are necessary to show if transmitters involved in eNOS-mediated penile erection, such as acetylcholine and VIP, which are also shown as activators of Akt/PKB kinases and MAP kinase 1/2 (Erk 1/2),^13,14,28 modulate eNOS activity by means of these kinases.

Besides eNOS regulation, both of the kinases investigated are also involved in various cell biological functions, such as regulation of signal transduction, apoptosis, cell attachment and cell proliferation.^7,29,30 Akt/PKB and MAP kinase 1/2 (Erk 1/2) surely play various roles in cavernosal tissue performance independent of eNOS regulation. The distinct expression and activation stage of the kinases in non-eNOS-containing structures, such as nerve fibres and vascular smooth muscle, gives substantial evidence for further functions carried out by these kinases in cavernosal regulation and maintenance.

Conclusion

This study focused on recently recognized upstream regulators of eNOS. It was shown that MAP kinase 1/2 (Erk 1/2) and serine/threonine specific protein kinase Akt/PKB are present and activated in the corpus cavernosum. These enzymes seem to be important for the achievement of erection. There was a distinct difference in the activation stage of the MAP kinase 1/2 (Erk 1/2) between endothelium and smooth muscle cells. This finding gives evidence for a cell-type-specific regulation of the eNOS-dependent NO release.

Further studies are required to show the functional input of these kinases and whether or not drugs affecting these pathways might be useful in the treatment of erectile dysfunction.

Furthermore, the study showed that all specimens from impotent patients with various primary diseases exhibited a greater amount of active MAP kinase 1/2 (Erk 1/2) in the cavernosal smooth muscle of fibromuscular stroma than in potent patients. In the future, further investigations should focus on the MAP Kinase 1/2 (Erk 1/2) expression, the various states of activation and their influence on the NO pathway.

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Figure 1 Homogenised corpus cavernosum from a potent man was electrophoresed on a 12.5% SDS gel, blotted and treated with antibodies against the MAP kinase 1/2 (ERK) and Akt/PKB (Akt) and the activated form of the Akt/PKB (p-Akt) and the activated form of the MAP Kinase 1/2 (p-ERK). The non-phosphorylated and phosphorylated (activated) MAP kinases 1/2 are shown at 42/44 kD and the non-phosphorylated and phosphorylated (activated) Akt/PKB are shown at 60 kD lane. The first lane (marker) shows the marker (BOA, Biomol, Hamburg, Germany). While Akt/PKB was mainly found in an active stage (p-Akt), only the MAP kinase p42 was mainly activated, while the activation of MAP kinases p44 was weak (p-ERK).

Figure 2 Sections of human corpus cavernosum tissue from potent men (potent) immunostained by antibodies for Akt/PKB (A) and for the phosphorylated (activated) Akt/PKB (B). (A) The endothelium (arrowheads) of the cavernosal sinus (S) and the smooth musculature (M) of the fibromuscular stroma are stained by the Akt/PKB antibody. (B) Using an antibody which detects only the phosphorylated (activated) Akt/PKB the distinct activation stage of the enzyme is shown. Besides the endothelial location (arrowheads) of phosphorylated (activated) Akt/PKB, a distinct staining for the phosphorylated (activated) enzyme was found in the smooth muscle (M) of the cavernosal fibromuscular stroma. Bar=30 µm.

Figure 3 Light microscopic immunohistochemical detection of MAP Kinase 1/2 (Erk 1/2) (A) and phosphorylated (activated) MAP Kinase 1/2 (Erk 1/2 (B) in sections of human corpus cavernosum tissue from potent men (Potent). (A) The endothelium (arrowheads) of the cavernosal sinus (S) and smooth muscle cells (M) show a distinct expression of MAP kinase 1/2 (Erk 1/2). Only a small amount of MAP Kinase 1/2 was found in a phosphorylated (activated) stage in the smooth muscle cells (M) of the corpus cavernosum of a potent man, while a higher stage of activation was detected in the sinus endothelium (arrowheads). Bar=30 µm.

Figure 4 Endothelial cells and smooth muscle cells from small arteries in the fibromuscular stroma of the human corpus cavernosum (A-D) from potent men (potent). There is an intense colouring of the endothelium and weaker staining of the smooth muscle cells of a corporal artery by Akt/PKB (A), by phosphorylated (activated) Akt/PKB (C), by MAP Kinase 1/2 (Erk 1/2) (B) and by phosphorylated (activated) MAP Kinase 1/2 (Erk 1/2) (D). Phosphorylated (activated) Akt/PKB positive fine nerve fibres (arrows E) and phosphorylated (activated) MAP kinase 1/2 (Erk 1/2) positive fine nerve fibres (arrows F) are found in fibromuscular stroma. Bar A-D=30 µm, E-F=45 µm.

Figure 5 Photomicrographs of the human corpus cavernosum from patients with erectile dysfunction (ED) immunostained by antibodies for Akt/PKB (A), for phosphorylated (activated) Akt/PKB (C), for MAP kinase 1/2 (Erk 1/2) (B) and for phosphorylated (activated) MAP kinase 1/2 (Erk 1/2) (D). Distinct immunostaining for Akt/PKB and phosphorylated (activated) Akt/PKB as well as MAP kinase 1/2 (Erk 1/2) and phosphorylated (activated) MAP kinase 1/2 (Erk 1/2) is found in the endothelium bordering the cavernosal sinus (S). The smooth muscle cells (M) also show expression and phosphorylation (activation) of the AKT and MAP kinase 1/2 (Erk 1/2). Especially the MAP kinase 1/2 (Erk 1/2) was highly phosphorylated (activated) in the smooth muscle cells from patients with erectile dysfunction. Bar=45 µm.

Figure 6 The schema shows the suggested role of Akt/PKB and MAP kinases 1/2 for eNOS regulation in human penile erection.

Table 1 Patient demographics