MAP Kinase 1/2 (Erk 1/2) and Serine/Threonine Specific Protein

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MAP kinase 1=2 (Erk 1=2) and serine=threonine speciﬁc protein kinase Akt=PKB expression and activity in the human corpus cavernosum

F Sommer1*, T Klotz1 D Steinritz2, A Schmidt2, K Addicks2, U Engelmann1 and W Bloch2

1Department of Urology, University Medical Centre of Cologne, Cologne, Germany; and 2Institute I of Anatomy, University Medical Centre of Cologne, Cologne, Germany

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

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

Introduction 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) Penile erection is a hemodynamic process, invol- and serine=threonine specific protein kinase ving increased arterial inflow and restricted venous Akt=PKB.6,7 It has been demonstrated that the outflow, in coordination with corpus cavernosum serine=threonine specific protein kinase Akt=PKB 1 smooth muscle relaxation. Although this process is enzyme plays an important role in the activation of generally accepted to be under neuroregulatory eNOS. Mimicking the phosphorylation of Ser 1177 2 control, biochemical mediators released locally directly enhances enzyme activity and alters the from the endothelium and=or smooth muscle also sensitivity of the enzyme to Ca2þ, rendering max- 3 participate in initiating and maintaining erection. imum enzyme activity at sub-physiological concen- Nitric oxide (NO), which is produced both in trations of Ca2þ. Thus, phosphorylation of eNOS by cavernosal nerves and the endothelium, has been serine=threonine specific protein kinase Akt=PKB recognized as playing a key role in the physiology of represents a novel Ca2þ-independent regulatory 2 penile erection. Additionally, it has been shown mechanism for the activation of eNOS. Constitu- that endothelial NO synthase (eNOS) is one of the tively active serine=threonine specific protein ki- main sources of NO in the cavernosal tissue, which nase Akt=PKB stimulates the phosphorylation of is then available in endothelial cells and cavernosal eNOS, but the inactive kinase does not. As only a 4 smooth muscle cells. Nitrinergic innervation and few substrates for the serine=threonine specific eNOS expression have shown a broad heterogeneity; protein kinase Akt=PKB have been described so up until now, no correlation between eNOS expres- far, the results reported identify eNOS as a novel Akt 5 sion and erectile function has been observed. target.6 While active Akt=PKB increases the eNOS- mediated NO release, the activation of MAP Kinase *Correspondence: F Sommer, Klinik und Poliklinik fu¨ r 1=2 (Erk 1=2) leads to a reduction of eNOS activity Urologie der Universita¨tzuKo¨ln, Joseph-Stelzmann-Str. 9, through an as yet unknown phosphorylation pro- D-50924 Ko¨ln, Germany. E-mail: [email protected] cess. A direct interaction between these kinases Received 1 June 2001; revised 21 November 2001; seems unlikely because the inactivation of the accepted 29 January 2002 putative Akt=PKB phosphorylation state does not MAP kinase 1=2 and serine=threonine kinase F Sommer et al 218 block eNOS phosphorylation by MAP Kinase 1=2 pus cavernosum. Three patients underwent trans- (Erk 1=2).7 NO production in endothelial cells can sexual operations (male – female). These patients be a response to different stimuli, such as acetylcho- had undergone a long-term hormonal pre-treatment, line,8 shear stress, bradykinin, substance P and but reported normal erections (Table 1). 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 Immunohistochemistry Acetylcholine, one of the key transmitters for the 11 erectile function, has been shown to act as a Prior to immunohistochemical examination, tissue relaxant stimulus in the corpora cavernosa of rats, sections (6 mm) were placed in a bathing solution of by means of activation of nNOS and eNOS-derived 3% H O and methanol for 20 min, then permeabi- 12 2 2 NO release. Other researchers were able to demon- lized for 10 min with 0.25% Triton-X in 0.1 M TBS. strate acetylcholine as an activator of Akt=PKB and The sections were then treated with 5% bovine 13,14 MAP Kinase 1=2 (Erk 1=2). Thus, we considered serum albumin (BSA) solution in TBS. Before each Akt=PKB and MAP Kinase 1=2 (Erk 1=2) as possible step, the sections were rinsed three times in TBS upstream regulators of eNOS in the corpus caverno- buffer. Incubation with the primary antibody oc- sum, depending on the expression and distribution curred in a TBS-based solution of 0.8% BSA for 24 h of these enzymes. at 4C. The anti-Erk 1=2 antibody was applied in a Up to now, cavernosal tissue has not been dilution of 1:400 and the anti-Akt kinase antibody examined for the MAP Kinase 1=2 (Erk 1=2) and was applied in a dilution of 1:150. The antibodies the serine=threonine specific protein kinase for the phosphorylated (activated) forms of the Akt=PKB enzymes. The aim of this study was to enzymes were applied in a dilution of 1:400 (for investigate the distribution and basal activation activated Map Kinase 1=2 (for Erk 1=2) and 1:500 stage of the MAP Kinase 1=2 (Erk 1=2) and the (activated AKT=PKB). After rinsing with TBS, the serine=threonine specific protein kinase Akt=PKB in sections were incubated with the corresponding the human corpus cavernosum. In this context, we secondary biotinylated antibody for 1 h at room examined cavernosal tissues from both potent and temperature. A streptavidin – horseradish peroxi- impotent patients, in order to elucidate possible dase complex was then applied as a detection differences between these groups. system (1:150 dilution) for 1 h. Finally, the staining

Table 1 Patient demographics Patients and methods Age, when No. of operated History patient (years) of ED Patients and collection of tissue 1 54 None Congenital penile deviation 2 32 None Congenital penile deviation Twenty-four human corpus cavernosum tissue spe- 3 60 None Congenital penile deviation cimens were obtained from patients subjected to 4 36 None Congenital penile deviation 5 43 None Congenital penile deviation penile surgery, with the patients’ informed consent. 6 63 None Penile deviation The specimens were immediately fixed in 4% 7 16 None Congenital penile deviation paraformaldehyde for 4 h and then rinsed in 0.1 M 8 26 None Congenital penile deviation phosphate-buffered saline (PBS) for 24 h. The tissue 9 36 None Penile injury was stored for 12 consecutive hours in a PBS 10 63 None Penile deviation 11 32 None Congenital penile deviation solution containing 18% sucrose for cryoprotection 12 45 None Congenital penile deviation and then frozen to 7 80 C. 13 39 None Congenital penile deviation Fourteen of the patients (age range 16 – 63 y; mean 14 40 None Congenital penile deviation age 42 y) with normal erectile function suffered from 15 41 Yes Arteriel insuffiency 16 29 Yes Insuffiency of the veno-occlusive penile deviations. All patients were treated via system Nesbit’s surgical procedure. Normal erectile func- 17 53 Yes Arteriel insuffiency tion was ascertained by anamnestic evaluation. 18 66 Yes Insuffiency of the veno-occlusive Seven patients (age range 27 – 66 y; mean age 46 y) system had shown complete erectile dysfunction for more 19 27 Yes Insuffiency of the veno-occlusive system than 2 y. This group underwent implantation of 20 58 Yes Radical cystectomy flexible hydraulic penile prostheses. Four patients 21 50 Yes Insuffiency of the veno-occlusive suffered from severe venous leakage, and one system patient from impotence due to radical surgery of a 22 37 None Transsexualism 23 38 None Transsexualism bladder carcinoma. Two patients had diabetes and a 24 27 None Transsexualism combined arterial-venous insufficiency of the cor-

International Journal of Impotence Research MAP kinase 1=2 and serine=threonine kinase F Sommer et al 219 was developed for 3 – 10 min with 150 ml 3,3-diami- kinase 1=2 [ERK 1=2] (1:1200, Upstate Biotechnol- nobenzidine tetrahydrochloride (DAB), 150 ml ogy), mouse anti-MAP Kinase, activated [Diphos- NH4Cl, 50 ml glucose oxidase, 300 ml 10% glucose, phorylated ERK 1=2] (1:1000, Sigma, Saint Louis, and 300 ml NiSO4 in 15 ml phosphate buffer (PB). MO, USA), goat anti-rabbit conjugated biotin (1:400, Negative control sections were incubated without Dako, Glostrup, Denmark), goat anti-mouse conju- the primary antibody. gated biotin (1:400, Dako) and streptavidin- conjugated HRP (1:150, Amersham, Life Science, Bucks, UK) were used. Immunoblot Results

Homogenisation. Human corpus cavernosum was Immunoblot analysis obtained fresh from the operating room and frozen in fluid nitrogen. Tissue was homogenised and treated with dissection buffer pH 7.4 (240 mM The immunoblot analysis reveals that the MAP sucrose, 1 mM PMSF, 20 mM PIPES, 10 mM EDTA, kinase 1=2 (Erk 1=2) and serine=threonine specific 50 mM NaH2PO4). Protein concentration was deter- protein kinase Akt=PKB are expressed in human mined according to Bradford.15 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 SDS – PAGE, immunoblotting and detection. SDS – corpus cavernosum. Additionally, it was demon- PAGE was performed according to Laemmli.16 strated that active MAP Kinase 1 (Erk 1) is present in Protein samples were denatured by boiling in 0.8% greater quantity than active MAP Kinase 2 (Erk 2). SDS and 0.5% b-mercaptoethanol (v=v) and electro- Inactive MAP Kinase 1=2 (Erk 1=2) has a similar phoresed in a 12.5% gel. For immunoblotting, a expression level (Figure 1). semi-dry system (SemiDryBlotter II, KEMENTEC, Copenhagen, Denmark) was used. Proteins were blotted on a PVDF membrane (0.2 micron, BioRad Immunohistochemistry Laboratories, Munich, Germany) for 1 h at 170 mA. The membrane was blocked with 1% powdered To identify the cellular localization of the Akt=PKB milk in buffer I pH 7.8 (25 mM Tris=HCl, 137 mM and MAP Kinase, as well as their activation stage, in NaCl, 3 mM KCl, 0.5% Tween 20) for 1 h at RT (room temperature). The primary antibodies, rabbit anti- phospho-AKT-1=PKB a [Thr 308] (1:1000), rabbit anti-AKT-1=PKB a (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 4C. After washing (3615 min.) with buffer I, the relevant secondary antibodies were used for 1 h at room temperature (1:800, in buffer I þ 0.1% powdered milk). Following washing, steps were performed before incubation with streptavidin-conjugated HRP (1:300, Amersham, in buffer I þ 0.1% powdered milk) for 1 h at RT. After washing (3615 min.) with buffer II pH 7.8 (buffer I with 0.05% Tween 20), the detection was prepared with DAB solution. 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 Materials 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 The primary antibodies, rabbit anti-phospho-AKT- (activated) Akt=PKB are shown at 60 kD lane. The first lane = (marker) shows the marker (BOA, Biomol, Hamburg, Germany). 1 PKB a [Thr 308] (1:1000, Upstate Biotechnology, While Akt=PKB was mainly found in an active stage (p-Akt), only Lake Placid, NY, USA), rabbit anti-AKT-1=PKB a the MAP kinase p42 was mainly activated, while the activation of (1:400, Upstate Biotechnology), rabbit anti-MAP MAP kinases p44 was weak (p-ERK).

International Journal of Impotence Research MAP kinase 1=2 and serine=threonine kinase F Sommer et al 220 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 – 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 – 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 (Figure 3B). Akt=PKB and MAP kinase were also found in Figure 2 Sections of human corpus cavernosum tissue from cavernosal nerve fibres; here, the immunoreaction potent men (potent) immunostained by antibodies for Akt=PKB for both activated kinases was high (Figure 4E and (A) and for the phosphorylated (activated) Akt=PKB (B). (A) The F). endothelium (arrowheads) of the cavernosal sinus (S) and the No differences between potent and impotent smooth musculature (M) of the fibromuscular stroma are stained patients could be discovered for Akt=PKB (Figures by the Akt=PKB antibody. (B) Using an antibody which detects only the phosphorylated (activated) Akt=PKB the distinct activa- 2A and 5A) and MAP kinase 1=2 (Erk 1=2) (Figures tion stage of the enzyme is shown. Besides the endothelial 3A and 5B) in the endothelium and cavernosal and location (arrowheads) of phosphorylated (activated) Akt=PKB, a vascular smooth muscle. All specimens from im- distinct staining for the phosphorylated (activated) enzyme was potent patients with various primary diseases found in the smooth muscle (M) of the cavernosal fibromuscular showed a higher amount of the active MAP kinase stroma. Bar ¼ 30 mm. 1=2 (Erk 1=2) in the smooth muscle of cavernosal fibromuscular stroma than in potent patients (Figure NO activates soluble guanylate cyclase, which leads 5D), while no differences could be observed for the to the production of cyclic GMP (cGMP). cGMP active Akt=PKB (Figure 5C). signals via three different receptors in eukaryotic cells, including ion channels, phosphodiesterases, and protein kinases.19 Discussion There is convincing evidence that during erection the local release of NO and=or related factors produces relaxation of the human corpus caverno- Penile erection is produced by an increased blood sum.2 Non-adrenergic, non-cholinergic (NANC) flow to the corpus cavernosum (CC), made possible nerve-mediated NO release appears to be the most by the opening of penile resistance vessels (helicine important factor with respect to cavernosal smooth arteries), the relaxation of the CC cells, and occlu- muscle relaxation.17 NO has the capacity to activate sion of the venous outflow.1 The erectile response in guanylate cyclase. Submicromolar concentrations of several animal models depends on NO, produced by NO cause rapid and robust increases in cGMP levels nerves as well as by the vascular endothelium.1,17,18 in target cells.20 Changes in the cellular cGMP level

International Journal of Impotence Research MAP kinase 1=2 and serine=threonine kinase F Sommer et al 221 cavernosal smooth muscle relaxation in response to endothelium-mediated stimuli in hypercholestero- lemic 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 Figure 3 Light microscopic immunohistochemical detection of 23 – 25 MAP Kinase 1=2 (Erk 1=2) (A) and phosphorylated (activated) protein kinase Akt=PKB mediates the activa- MAP Kinase 1=2 (Erk 1=2 (B) in sections of human corpus tion of eNOS, leading to increased NO production. cavernosum tissue from potent men (Potent). (A) The endothe- Inhibition of the phosphatidylinositol-3-OH lium (arrowheads) of the cavernosal sinus (S) and smooth muscle kinase=Akt pathway or mutation of the Akt side of cells (M) show a distinct expression of MAP kinase 1=2 (Erk 1=2). the eNOS protein (at serine 1177) attenuates the Only a small amount of MAP Kinase 1=2 was found in a phosphorylated (activated) stage in the smooth muscle cells (M) serine phosphorylation and prevents the activation of the corpus cavernosum of a potent man, while a higher stage of of eNOS. Mimicking the phosphorylation of activation was detected in the sinus endothelium (arrowheads). Ser 1177 directly enhances enzyme activity and Bar ¼ 30 mm. alters the sensitivity of the enzyme to Ca2þ, rendering maximum enzyme activity at sub-physiological concentrations of Ca2þ. Thus, phosphoryla- as a secondary messenger lead to a physiological tion of eNOS by Akt=PKB represents a novel Ca2þ- response in these target cells. independent regulatory mechanism for activation of Smooth muscle relaxation is one of the most eNOS.6 important effects of the NO=cGMP pathway with It has been demonstrated that over-expression of regard to erectile function.5 Recently it has been the constitutively active Akt=PKB results in a 1.4- shown that endothelial NO synthase (eNOS) is one fold increase in intracellular cGMP, indicating that of the most important sources of NO in cavernosal eNOS can be activated by an Akt=PKB-dependent smooth muscle cells.4,21 Furthermore, it has been pathway.6 demonstrated that erectile dysfunction cannot be The involvement of serine=threonine specific reduced solely to pathological findings in penile NO protein kinase Akt=PKB in eNOS phosphorylation synthases, since no correlation was found between has been effectively established in several indepen- the expression of the NO synthases and erectile dent studies.6,26,27 Constitutively active seri- function in the cavernosal tissue of potent and ne=threonine specific protein kinase Akt=PKB impotent patients.4 However, a functional impair- stimulates the phosphorylation of eNOS, but the ment of eNOS activity may lead to impairment of inactive kinase does not. As only a few substrates for

International Journal of Impotence Research MAP kinase 1=2 and serine=threonine kinase F Sommer et al 222

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 ¼ 30 mm, E – F ¼ 45 mm.

the serine=threonine specific protein kinase muscle are the source of eNOS-mediated NO release Akt=PKB have been described so far, the results in the corpus cavernosum.5 So it can be speculated reported identify eNOS as a novel Akt target.6 Our that the activation of eNOS by serine=threonine study showed endothelial and smooth muscle specific protein kinase Akt=PKB in the corpus expression of the serine=threonine specific protein cavernosum leads to increased NO production, kinase Akt=PKB. Active serine=threonine specific resulting in smooth muscle relaxation with conse- protein kinase Akt=PKB was found in endothelium quent erection (Figure 6). The finding of a distinct and cavernosal and vascular smooth muscle cells. amount of active serine=threonine specific protein The sinus endothelium and the cavernosal smooth kinase Akt=PKB in cavernosal smooth muscle cells

International Journal of Impotence Research MAP kinase 1=2 and serine=threonine kinase F Sommer et al 223

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 ¼ 45 mm. and the sinus endothelium hints at a possibly major and smooth muscle expression of the MAP kinase role played by the NO pathway in achieving 1=2 (Erk 1=2). This result makes it likely that MAP erection. kinase 1=2 (Erk 1=2) plays a role as negative eNOS is reversibly associated with protein ki- regulator of eNOS activity in the cavernosal tissue nases that are implicated in opposing regulatory (Figure 6). effects on the enzyme, leading to both eNOS In our study, we did not find any distinct activation (Akt) and inhibition (Erk).7 differences between potent and impotent patients There is evidence for the direct involvement of with regard to the expression and distribution of MAP Kinase 1=2 (Erk 1=2) in eNOS regulation. MAP kinase 1=2 (Erk 1=2). However, the specimens Showing that eNOS can be phosphorylated (acti- from impotent patients with various primary dis- vated) by MAP kinase 1=2 (Erk 1=2) seems highly eases showed a higher expression of the active MAP likely, since several additional residues in eNOS kinase 1=2 (Erk 1=2) in the smooth muscle of undergo phosphorylation in endothelial cells. This cavernosal fibromuscular stroma under the observed suggests that diverse kinase pathways might have an basal conditions than in potent patients. It can be important influence on the enzyme. Studies by speculated that the higher activation stage of MAP Bernier et al. provided evidence indicating that kinase 1=2 (Erk 1=2) in cavernosal smooth muscle of MAP kinase 1=2 (Erk 1=2) plays a key role in eNOS impotent patients impairs the switch-on of eNOS by regulation.7 It was strongly suggested that phos- erectile stimulation. This could identify a possible phorylation of eNOS catalysed by MAP kinase 1=2 pathway affecting erectile dysfunction. (Erk 1=2) can lead to enzyme inhibition, and it was We have to stress the limitations of our study shown that in vitro phosphorylation of eNOS by with respect to the small number of patients, which MAP kinase 1=2 (Erk 1=2) is associated with a did not allow for a statistical analysis. Additionally, reduction in enzyme activity. MAP kinase 1=2 (Erk it should be stressed that our study shows the 1=2) inhibits eNOS by phosphorylating the enzyme expression or distribution of the kinases, but only in endothelial cells.7 Our study showed endothelial the basal functional status. Further studies are

International Journal of Impotence Research MAP kinase 1=2 and serine=threonine kinase F Sommer et al 224 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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