Stefan Ückert

CHARACTERIZATION AND FUNCTIONAL RELEVANCE OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ISOENZYMES OF THE HUMAN PROSTATE

PURPOSE Current pharmacological concepts in the treatment of benign prostatic hyperplasia are aimed at the static and dynamic component of the disease. Mainly alpha-blocking agents are used to reduce the adrenergic tone of prostatic myocytes, thus, increasing urine flow through the prostatic urethra by reducing urethral resistance. With the introduction of the phosphodiesterase (PDE) 5 inhibitor sildenafil the concept of PDE inhibition has gained tremendous interest in the field of urology. We characterized PDE isoenzymes from human prostatic tissue by molecular biology and protein chemistry, and investigated the effects of various PDE inhibitors compared with standard pharmacological agents on adrenergic tension in isolated human prostatic strips. MATERIALS AND METHODS PDE isoenzymes were characterized by reverse transcriptase-polymerase chain reaction and anion exchange chromatography. Using the organ bath technique we determined the relaxing effects of the PDE inhibitors papaverine, vinpocetine (Biomol GmbH, Hamburg, Germany), erythro-9-[2-hydrox-3-nonyl]adenine hydrochloride (Tocris Cookson, Ltd., Bristol, United Kingdom) rolipram (Schering AG, Berlin, Germany), zaprinast (Rhône-Poulenc Rorer, Ltd., Dagenham, United Kingdom) and sildenafil (Pfizer, Ltd., Sandwich, United Kingdom), the adenylate cyclase activating agent forskolin (ICN Biomedicals, Aurora, Ohio) and the nitric oxide donor sodium nitroprusside on adrenergic tension in prostatic strips isolated from the transition zone. RESULTS Reverse transcriptase-polymerase chain reaction revealed messenger RNA transcripts encoding for PDE 1, 2, 4, 5, 7, 8, 9 and 10 in the various anatomical regions of the human prostate, including the peripheral, central and transition zones. Except for complementary DNA encoding for PDE 1C, complementary DNA fragments encoding for PDE 1A, 1B, 2A, 4A, 4B, 4C, 4D, 5A, 7A, 8A, 9A and 10A were found at almost even ratios in the different histological regions of the prostate. The hydrolytic activities of PDE 4 and 5 were present in the cytosolic fraction of prostatic tissue, whereas in the particulate fraction only the hydrolytic activity of PDE 4 was detected. Adrenergic tension in prostatic strip preparations was reversed by forskolin, sodium nitroprusside, and inhibitors of PDE 4 and 5. CONCLUSIONS Our results demonstrate the presence and functional relevance of PDE isoenzymes 4 and 5 in human prostatic tissue. These studies support the possible use of inhibitors of PDE 4 and 5 for treating urinary obstruction secondary to benign prostatic hyperplasia.

Anatomy, Physiology, and Pathophysiology of Erectile Dysfunction

INTRODUCTION Significant scientific advances during the past 3 decades have deepened our understanding of the physiology and pathophysiology of penile erection. A critical evaluation of the current state of knowledge is essential to provide perspective for future research and development of new therapies. AIM To develop an evidence-based, state-of-the-art consensus report on the anatomy, physiology, and pathophysiology of erectile dysfunction (ED). METHODS Consensus process over a period of 16 months, representing the opinions of 12 experts from seven countries. MAIN OUTCOME MEASURE Expert opinion was based on the grading of scientific and evidence-based medical literature, internal committee discussion, public presentation, and debate. RESULTS ED occurs from multifaceted, complex mechanisms that can involve disruptions in neural, vascular, and hormonal signaling. Research on central neural regulation of penile erection is progressing rapidly with the identification of key neurotransmitters and the association of neural structures with both spinal and supraspinal pathways that regulate sexual function. In parallel to advances in cardiovascular physiology, the most extensive efforts in the physiology of penile erection have focused on elucidating mechanisms that regulate the functions of the endothelium and vascular smooth muscle of the corpus cavernosum. Major health concerns such as atherosclerosis, hyperlipidemia, hypertension, diabetes, and metabolic syndrome (MetS) have become well integrated into the investigation of ED. CONCLUSIONS Despite the efficacy of current therapies, they remain insufficient to address growing patient populations, such as those with diabetes and MetS. In addition, increasing awareness of the adverse side effects of commonly prescribed medications on sexual function provides a rationale for developing new treatment strategies that minimize the likelihood of causing sexual dysfunction. Many basic questions with regard to erectile function remain unanswered and further laboratory and clinical studies are necessary.

Phosphodiesterase 1 inhibition in the treatment of lower urinary tract dysfunction: from bench to bedside.

Abstract Anticholinergic drugs are currently the therapy of choice to treat urgency and urge incontinence. However, muscarinergic receptor blockers with adequate selectivity for detrusor smooth muscle are not available. Also, in contrast to the normal detrusor, the unstable detrusor neurotransmission seems to be at least partially regulated by non-cholinergic (NANC) pathways. These factors may explain the common side effects and the limited clinical efficacy of these compounds. Specific modulation of intracellular second messenger pathways offers the possibility of organ selective manipulation of tissue function, specifically contraction and relaxation of smooth musculature. Because of their central role in the intracellular regulation of smooth muscle tone phosphodiesterases (PDEs) are an attractive pharmacological targets. The PDE 5 specific inhibitor sildenafil (Viagra) has revolutionized the treatment of patients with erectile dysfunction. Numerous other PDE inhibitors are currently under investigation for the treatment of various disorders. We investigated the role of PDEs in human detrusor smooth muscle. Our data demonstrate the presence of five PDE isoenzymes in human detrusor and suggest, for the first time, that the cAMP pathway and the calcium/calmodulin-stimulated PDE (PDE 1) are of functional importance in the intracellular regulation in this tissue in vitro. In addition, initial clinical data with the PDE 1 inhibitor vinpocetine in patients not responding to standard anticholinergic therapy indicate a possible role for vinpocetine in the treatment of urgency, urge incontinence and, possibly, low compliance bladder and interstitial cystitis. The results of a larger randomized, double-blind, placebo-controlled, multicenter trial with vinpocetine show a tendency in favor of vinpocetine over placebo; however, statistically significant results were documented for one parameter only. This might be due to the rather low dosage chosen and the small sample size. Further studies are necessary and currently underway to delineate the optimal dosage, indications and patient population. Modulation of intracellular key enzymes effecting second messenger metabolism, i.e. isoenzyme-selective PDE inhibition is a novel approach which possibly avoids the limitations of anticholinergic therapy in patients with lower urinary tract dysfunction.

Cyclic nucleotide phosphodiesterase (PDE) isoenzymes in the human detrusor smooth muscle

Phosphodiesterases (PDEs) are key enzymes involved in the regulation of intracellular cyclic nucleotide metabolism. The aim of the present study was to identify and to characterize the PDE isoenzymes present in the human detrusor smooth muscle. Human detrusor PDE isoenzymes were separated by Q-Sepharose anion exchange and calmodulin-agarose affinity chromatography and characterized upon their kinetic characteristics and their sensitivity to allosteric modulators and inhibitors. All five presently known PDE isoenzyme families were identified: one high-affinity, low-Km calcium/calmodulin-stimulated PDE I with a slight preference for cGMP over cAMP, one cGMP-stimulated PDE II, one cGMP-inhibited PDE III, one cAMP-specific PDE IV and one cGMP-specific PDE IV. All five known PDE isoenzyme families exist in human detrusor smooth musculature. The kinetic characteristics, together with functional in vitro studies, suggest that the PDE I may be of importance in the intracellular regulation of the human detrusor smooth muscle tone.

Possible role of bradykinin and angiotensin II in the regulation of penile erection and detumescence

OBJECTIVES To examine the functional effects of bradykinin (BK) and angiotensin II (AN II) on isolated human cavernous tissue and to detect any changes in the AN II levels in cavernous and peripheral blood samples taken from healthy volunteers at different functional conditions of the penile erectile tissue. Metabolites of the renin-angiotensin system and endothelium-derived vasoactive substances are known to be involved in the regulation of arterial vascular tone. The human corpus cavernosum (HCC), consisting of endothelial and smooth muscle cells, can be regarded as a compartment comparable to the vascular system. METHODS The relaxing and contracting properties of BK and AN II on isolated HCC were investigated using the organ bath technique. Tissue levels of adenosine-3,5-cyclic monophosphate (cAMP) and guanosine-3,5-cyclic monophosphate (cGMP) were determined using specific radioimmunoassays, after exposing isolated HCC strips in a dose-dependent manner to BK, forskolin, and sodium nitroprusside. Blood samples were drawn simultaneously from the corpus cavernosum and cubital vein of 34 healthy volunteers at stages of penile flaccidity, tumescence, rigidity, and detumescence. Penile erection was induced by audiovisual and tactile stimulation. AN II levels were determined using a radioimmunoassay. RESULTS In vitro, BK, forskolin, and sodium nitroprusside elicited dose-dependent relaxation of norepinephrine-induced tension of isolated HCC, and AN II evoked dose-dependent contraction of the HCC strips. The relaxing potency of BK was paralleled by its ability to elevate the intracellular levels of cAMP and cGMP. In vivo, the AN II levels in the cavernous plasma increased from 21.8 +/- 4.6 pg/mL in the flaccidity phase to 27.9 +/- 10 pg/mL in the detumescence phase. In the peripheral plasma, the AN II levels were 17.2 +/- 6.2 to 19.5 +/- 6.5 pg/mL in the respective penile stages. Thus, the mean AN II levels in the cavernous blood were about 30% higher than in the blood samples taken from the cubital vein. In the cavernous blood, the increase in the AN II plasma levels in the detumescence phase (27.9 +/- 10 pg/mL) was statistically significant. CONCLUSIONS Our results suggest that penile cavernous smooth muscle tone is partially balanced by kinin-induced relaxation and AN II-induced contraction. Since the tissue and plasma levels of both peptides are regulated by the activity of the angiotensin-converting enzyme, there might be a rationale for the use of angiotensin-converting enzyme inhibitors in the treatment of erectile dysfunction associated with arterial hypertension.

The Mechanism of Action of Phosphodiesterase Type 5 Inhibitors in the Treatment of Lower Urinary Tract Symptoms Related to Benign Prostatic Hyperplasia

CONTEXT Clinical trials of phosphodiesterase type 5 inhibitors (PDE5-Is) have consistently demonstrated a significant reduction in lower urinary tract symptoms (LUTS) and small urinary flow rate changes in men with benign prostatic hyperplasia (BPH). OBJECTIVE This review presents the proposed mechanisms of action of PDE5-Is in the treatment of BPH-LUTS focusing on the localization of PDE5 isoenzymes in the pelvic structures; smooth muscle relaxation in the bladder, prostate, and supporting vasculature; increased blood perfusion of the bladder and prostate; and modulation of sensory impulses from these organs. EVIDENCE ACQUISITION Literature describing in vitro, preclinical, or clinical studies of pathologic processes contributing to LUTS or effects of PDE5 inhibition on the lower urinary tract (LUT) was selected for review. EVIDENCE SYNTHESIS We objectively assessed and summarized the published data focusing on articles published within the past 10 yr. Articles before the time cut-off were included if historically relevant. CONCLUSIONS The PDE5 isoenzymes are highly expressed in the LUT including the bladder, prostate, and their supporting vasculature. In vitro assays have demonstrated PDE5-Is by regulating cyclic guanosine monophosphate (cGMP) degradation and enhancing the nitric oxide/cGMP signaling pathway to relax human smooth muscle strips from the prostate, bladder, and LUT arteries. In animals characterized by ischemia/hypoxia of the genitourinary tract, treatment with PDE5-Is increases bladder and prostate tissue oxygenation. PDE5-Is have been shown to reduce nonvoiding contractions and bladder afferent nerve firing in decerebrate spinal cord-injured rats, and to reduce mechanosensitive afferent activities of both Aδ- and C-fibers in an irritated or overextended bladder model.

Phosphodiesterase isoenzymes as pharmacological targets in the treatment of male erectile dysfunction

Abstract Based on the increasing knowledge of intracellular signal propagation in cavernous smooth muscle tone regulation, which is of major importance to the understanding of both the physiology of erection and the pathophysiology of erectile dysfunction, selective phosphodiesterase (PDE) inhibitors have recently been introduced in the treatment of erectile dysfunction. The first promising clinical data on the use of the orally active PDE5 inhibitor Sildenafil in the treatment of erectile dysfunction were accompanied by boosting research activities on cavernous intracellular signal transduction and phosphodiesterase characterization with the aid of molecular biology and protein chemistry. The presence of mRNA transcripts specific for 14 different human phosphodiesterase isoenzymes and isoforms in human cavernous tissue was shown by RT-PCR: Three isogenes of PDE1, PDE2A and 10A, which hydrolyse cAMP as well as cGMP, the cAMP-specific PDE3A, four isogenes of PDE4, PDE7A and PDE8A, as well as cGMP-specific PDEs PDE5A and PDE9A. Using anion exchange chromatography, the activities of PDE isoenzymes 2, 3, 4, and 5 were detected in cytosolic supernatants of human cavernous smooth muscle. To date, the efficacy and safety of several next generation PDE5 inhibitors for use in the treatment of male erectile dysfunction are under evaluation in vitro and in vivo. Further research will possibly allow identification of diagnostic tools for erectile dysfunction and of even more selective drugs in its therapy.

The nitric oxide pathway in the human prostate: clinical implications in men with lower urinary tract symptoms

To date, there is an increasing interest in the nitric oxide (NO) pathway as a potential pharmacological target to treat male lower urinary tract symptomatology (LUTS). In the transition zone of the human prostate, a dense nitrinergic innervation has been shown of the fibromuscular stroma, glandular epithelium and blood vessels. The expression of key proteins of the NO pathway, such as the endothelial and neuronal nitric oxide synthase (eNOS, nNOS), cGMP-degrading phosphodiesterase type 5 (PDE5) and cGMP-binding protein kinase (cGK), has also been demonstrated. The hypothesis that an impaired NO/cGMP-signaling may contribute to the pathophysiology of benign prostatic hyperplasia (BPH) is supported by the results from randomized, placebo-controlled clinical studies, indicating that NO donor drugs and PDE5-inhibitors sildenafil, tadalafil and vardenafil may be useful to treat storage and voiding dysfunctions resulting from LUTS in men. Thus, given a potential role of the NO-pathway in the prostate and/or in other parts of lower urinary tract (e.g. bladder), the enhancement of the NO signaling by NO donor drugs, PDE5 inhibitors or activators of the soluble guanylyl cyclase (sGC) may represent a new therapeutic strategy for the treatment of LUTS. This review serves to focus on the role of NO and the NO-dependent signaling in the control of smooth muscle function in the human prostate. Results from clinical trials in men with LUTS/BPH are also discussed.

Effects of Phosphodiesterase Inhibitors on Tension Induced by Norepinephrine and Accumulation of Cyclic Nucleotides in Isolated Human Prostatic Tissue

OBJECTIVES To further evaluate the mechanism of action of phosphodiesterase (PDE) inhibitors on the human prostate, the effects of PDE4 and PDE5 inhibitors on the tension induced by norepinephrine (NE) and on the intracellular levels of cyclic nucleotides in isolated human prostatic tissue were investigated. METHODS Using the organ bath technique, the effects of increasing concentrations (1 nM to 10 microM) of the PDE5 inhibitors sildenafil, tadalafil, and vardenafil and the PDE4 inhibitors rolipram and RP 73401 on the tension induced by NE (40 microM) of prostate strip preparations were investigated. The accumulation of cyclic guanosine monophosphate and cyclic adenosine monophosphate in response to drug exposure was determined by radioimmunoassays. RESULTS The tension induced by NE was dose dependently reversed by the drugs with the following rank order of efficacy: tadalafil greater than RP 73401 greater than rolipram greater than or equal to vardenafil greater than sildenafil. The maximal reversion of tension values ranged from 52.3% (tadalafil) to 17% (sildenafil). Of the PDE inhibitors, only tadalafil induced a 50% reversion of the initial tension. The most prominent enhancement in tissue cyclic adenosine monophosphate was registered in response to RP 73401 (11-fold), and cyclic guanosine monophosphate levels were significantly elevated by tadalafil, vardenafil, and sildenafil (28-fold, 12-fold, and 3-fold, respectively). CONCLUSIONS Our results have demonstrated that drugs interfering with the cyclic nucleotide-mediated pathways can reverse the tension induced by NE in isolated prostatic tissue and elevate cyclic adenosine monophosphate and cyclic guanosine monophosphate. Our findings serve to explain how PDE inhibitors can affect symptoms of lower urinary tract symptoms and benign prostatic hyperplasia.

Plasma levels of angiotensin II during different penile conditions in the cavernous and systemic blood of healthy men and patients with erectile dysfunction

OBJECTIVES To detect changes in plasma levels of angiotensin II (Ang II) under different functional conditions of the penile erectile tissue in the cavernous and systemic blood of patients with erectile dysfunction (ED) and compare them with the course of Ang II in healthy male subjects. It has been shown that the mammalian corpus cavernosum produces and secretes physiologically relevant amounts of the vasoconstrictive peptide Ang II and that intracavernosal injection of Ang II terminates penile erection in the dog. Thus, we speculated whether a dysregulation in the secretion or degradation of Ang II might contribute to the manifestation of ED. METHODS Thirty-four healthy adult men and 48 patients with ED of either organogenic or psychogenic etiology were exposed to visual and tactile erotic stimuli to elicit penile tumescence and, in the group of healthy subjects, rigidity. Whole blood was simultaneously aspirated from the corpus cavernosum and the cubital vein during the different functional conditions of the penis. Ang II plasma levels were measured using a radioimmunoassay. RESULTS In healthy men, the Ang II levels in the cavernous plasma increased from 22.1 +/- 7.1 pg/mL in the phase of penile rigidity to 27.9 +/- 10 pg/mL in the detumescence phase. In the peripheral plasma, the Ang II levels were 17.2 +/- 6.2 to 19.5 +/- 6.5 pg/mL over the respective penile stages. The courses of Ang II registered in the patients were similar to those detected in the healthy men. In the patients and healthy men, systemic Ang II levels were found to be lower than the concentrations detected in the cavernous blood. In the group of organogenic patients, the Ang II levels during penile flaccidity in the systemic and cavernous blood were higher than those registered in the blood samples taken from the healthy men. CONCLUSIONS Our results suggest that cavernous smooth muscle tone is, in part, balanced by Ang II-induced contraction and that Ang II might be involved in the initiation of penile detumescence in men. That Ang II plasma levels are generally elevated in the systemic and cavernous blood of patients with an organogenic etiology of ED may hint at the significance of Ang II in the pathophysiology of ED. Since the tissue and plasma levels of Ang II are regulated by the activity of angiotensin-converting enzyme, there might be a rationale for the use of angiotensin-converting enzyme inhibitors in the treatment of vasculogenic ED.