OBJECTIVESTo investigate the effects of antisense oligodeoxynucleotide(ASON) on the cyclic nucleotide monophosphates (cNMP) in smooth muscle cells of human corpus cavernosum, and provide experimental groundwork for the gene therapy of erectile dysfunction.

METHODSPDE5 gene ASON(containing exon 1) was transfected into the corpus cavernosum smooth muscle cells with the presence of liposome DOTAP. Another sense oligodeoxynucleotide(SON) and 1% of bovine serum were also transducted into the cells as controls. Two of cNMP, cAMP and cGMP, were probed and measured by ELISA at 1, 2, 4, 6, 10, 24 and 48 h after transfection.

RESULTSAfter transfection, the level of cGMP(1-6 h) in human corpus cavernosum smooth muscle cells was significantly higher than that in controls(P < 0.01).

CONCLUSIONSThe PDE5 gene ASON had been showed to manifest stimulative effect on the cGMP in smooth muscle cells of human corpus cavernosum in vitro, and it provides experimental groundwork for the gene therapy of erectile dysfunction.

3',5'-Cyclic-GMP Phosphodiesterases ; antagonists & inhibitors ; genetics ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Humans ; Male ; Muscle, Smooth ; drug effects ; metabolism ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Penis ; cytology

OBJECTIVETo investigate the effect of phosphodiesterase type 5 (PDE5) small interfering RNA (siRNA) on the cGMP in the smooth muscle cells of human corpus cavernosum, and to provide an experimental groundwork for the gene therapy of erectile dysfunction (ED).

METHODSSmall interfering RNAs targeting PDE5 gene were synthesized by using web design software provided by Ambion, three siRNAs and a control siRNA were synthesized by Ambion. siRNAs were transfected into the smooth muscle cells of human corpus cavernosum by using siPORT Lipid reagent. cGMP was detected by ELISA at different times (24, 48, 72 and 96 h) after transfection.

RESULTSThe cGMP levels of the siRNA1, siRNA2 and siRNA3 groups were significantly higher than those of the siRNA control and blank control groups (P < 0.05), and so was it in the siRNA1 group than the siRNA2 and siRNA3 groups (P < 0.05), with significant difference between the siRNA control and the blank control group (P > 0.05).

CONCLUSIONThe synthesized siRNAs in vitro are capable of increasing the level of cGMP in the smooth muscle cells of human corpus cavernosum, different siRNAs with different capabilities. The siRNA technique could provide not only an extremely powerful tool for the functional analysis of genome but also a new approach to ED gene therapy.

3',5'-Cyclic-GMP Phosphodiesterases ; genetics ; Cells, Cultured ; Cyclic GMP ; metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Gene Silencing ; Humans ; Male ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Penis ; metabolism ; RNA, Small Interfering ; pharmacology ; Transfection

OBJECTIVETo construct an antisense RNA recombinant adenovirus vector of the human PDE5A1 promoter gene.

METHODSA cDNA fragment containing the human PDE5A1 promoter and the human PDE5A1-specific exon was determined according to the gene bank. The antisense RNA fragment was synthesized artificially and subcloned into the pENTR. Then, the sequence of pENTR fragment was detected, and the recombinant adenovirus vector pAd/CMV/V5/antisense-PDE5A1 was constructed by LR reaction with the Gateway expression system. The identified recombinant adenovirus plasmid was digested with Pac I and transformed into 293A cells to package recombinant adenovirus particles. The recombinant adenovirus particles were tested with PCR technique and purified after acquisition by CsCl density gradient ultracentrifugation.

RESULTSThe sequencing result showed a 145 bp fragment in pENTR, which was proved to be the domain of the antisense RNA fragment. PCR confirmed that the antisense RNA fragment was cloned into the recombinant adenovirus vector pAd/CMV/V5/antisense-PDE5A1 successfully and could infect 293A cells. The titer of virus stocks was up to 10(8) - 10(10)/microl after purification.

CONCLUSIONWith the Gateway expression system, the culturing and reproduction of 293A cells can reproduce recombinant adenovirus pAd/CMV/V5-DEST successfully, and the recombinant adenovirus vector can meet the need of in vivo gene transfection in laboratory studies.

3',5'-Cyclic-GMP Phosphodiesterases ; genetics ; Adenoviridae ; genetics ; Cell Line ; Cloning, Molecular ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Genetic Vectors ; Humans ; Plasmids ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; RNA, Antisense ; genetics ; Recombination, Genetic

OBJECTIVESTo study the PDE5 activity in corpora cavernosa of the Ganyu Qizhi model penis and the effect of the Chinese herbal medicine Shugan Liqi Huoxue (SLH) ointment on it.

METHODSNon-injury stress stimulus method similar to human spirit stress was used to extablish the Ganyu Qizhi animal(rat) model, and the PDE5 activity in corpora cavernosa of the rat penis was measured by the method of immunohistochemistry and computer image analysis.

RESULTSThe PDE5 activity in corpora cavernosa of the high-dosage SLH group was significantly different from that of the model group (P < 0.01).

CONCLUSIONGanyu Qizhi may increase the PDE5 activity in corpora cavernosa of the penis while SLH can reduce such activity.

3',5'-Cyclic-GMP Phosphodiesterases ; metabolism ; Animals ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Drugs, Chinese Herbal ; pharmacology ; Erectile Dysfunction ; etiology ; Male ; Medicine, Chinese Traditional ; Models, Animal ; Penis ; drug effects ; enzymology ; Rats ; Rats, Sprague-Dawley

Current available treatment options for erectile dysfunction (ED) are effective but not without failure and/or side effects. Although the development of phosphodiesterase type 5 (PDE5) inhibitors (i.e. sildenafil, tadalafil and vardenafil) has revolutionized the treatment of ED, these oral medications require on-demand access and are not as effective in treating ED related to diabetic, post-prostatectomy and severe veno-occlusive disease states. Improvement in the treatment of ED is dependent on understanding the regulation of human corporal smooth muscle tone and on the identification of relevant molecular targets. Future ED therapies might consider the application of molecular technologies such as gene therapy. As a potential therapeutic tool, gene therapy might provide an effective and specific means for altering intracavernous pressure "on demand" without affecting resting penile function. However, the safety of gene therapy remains a major hurdle to overcome before being accepted as a mainstream treatment for ED. Gene therapy aims to cure the underlying conditions in ED, including fibrosis. Furthermore, gene therapy might help prolong the efficacy of the PDE5 inhibitors by improving penile nitric oxide bioactivity. It is feasible to apply gene therapy to the penis because of its location and accessibility, low penile circulatory flow in the flaccid state and the presence of endothelial lined (lacunar) spaces. This review provides a brief insight of the current role of gene therapy in the management of ED.
3',5'-Cyclic-GMP Phosphodiesterases ; antagonists & inhibitors ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Erectile Dysfunction ; drug therapy ; genetics ; therapy ; Gene Transfer Techniques ; Genetic Therapy ; adverse effects ; Humans ; Male ; Phosphodiesterase Inhibitors ; therapeutic use ; Vasodilator Agents ; therapeutic use

OBJECTIVETo further investigate the action mechanisms of berberine (Ber) and to assess the effects of Ber on the mRNA expression of phosphodiesterase type 5 (PDE5) in rat corpus cavernosum.

METHODSAfter incubating with Ber for 1 or 3 h respectively, we examined the levels of PDE5 mRNA by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThere were PDE5A1 and PDE5A2 mRNA expressions in the rat corpus cavernosum with PDE5A2 as the dominant isoform. Ber could obviously inhibit the mRNA expression of the two isoforms in the rat penis and bring on a pronounced decrease in PDE5A2 (P < 0.01).

CONCLUSIONThe present study indicates that the inhibitory effect of Ber on PDE5 mRNA expression, especially on PDE5A2, might account for its molecular mechanism for treating ED.

3',5'-Cyclic-GMP Phosphodiesterases ; biosynthesis ; genetics ; Animals ; Berberine ; pharmacology ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Male ; Penis ; drug effects ; metabolism ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction

Since the introduction of the phosphodiesterase type 5 (PDE-5) inhibitor sildenafil in 1998, there has been a fundamental change in the treatment of erectile dysfunction (ED). Sildenafil has already been used by over 20 million men in over 100 countries, with a death rate similar to that of general population. The success rate of sildenafil amounts to an average of over 80%, and sildenafil has become the first choice for patients with ED. The development of two new PDE-5 inhibitors, vardenafil and tadalafil, has added to the options for the treatment of ED. In this review, a comparison is made of the pharmcodynamics, pharmacokinetics and adverse reactions between the three PDE-5 inhibitors to assess their efficacy and safety.
3',5'-Cyclic-GMP Phosphodiesterases ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Erectile Dysfunction ; drug therapy ; Humans ; Male ; Phosphodiesterase Inhibitors ; adverse effects ; pharmacokinetics ; therapeutic use ; Phosphoric Diester Hydrolases ; physiology ; Piperazines ; therapeutic use ; Purines ; Sildenafil Citrate ; Sulfones

Erectile response is centrally and peripherally regulated by androgens. The original insights into the mechanisms of action of androgens were that androgens particularly exert effects on libido and that erections in response to erotic stimuli were relatively androgen-independent. It was shown that sexual functions in men required androgen levels at the low end of reference values of testosterone. So it seemed that testosterone was not useful treatment for men with erectile difficulties, particularly following the advent of the phosphodiesterase type 5 (PDE5) inhibitors. However, approximately 50% of those treated with PDE5 inhibitors discontinue their treatment. A number of recent developments shed new light on testosterone treatment of erectile dysfunction (ED) in aging men. (1) A recent insight is that, in contrast to younger men, elderly men might require higher levels of testosterone for normal sexual functioning. (2) Several studies have indicated that PDE5 inhibitors are not always sufficient to restore erectile potency in men, and that testosterone improves the therapeutical response to PDE5 inhibitors considerably. (3) There is growing insight that testosterone has profound effects on tissues of the penis involved in the mechanism of erection and that testosterone deficiency impairs the anatomical and physiological substrate of erectile capacity, reversible upon androgen replacement. The synthesis of PDE5 is upregulated by androgens, and the arterial inflow into the penis is improved by giving androgen. The above invites a re-examination of the merits of giving testosterone to aging men with ED. The beneficial effects of PDE5 inhibitors may only be optimally expressed in a eugonadal environment.
3',5'-Cyclic-GMP Phosphodiesterases ; Aging ; physiology ; Animals ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Humans ; Male ; Middle Aged ; Penile Erection ; drug effects ; physiology ; Penis ; anatomy & histology ; drug effects ; Phosphodiesterase Inhibitors ; pharmacology ; therapeutic use ; Phosphoric Diester Hydrolases ; physiology ; Piperazines ; therapeutic use ; Purines ; Sildenafil Citrate ; Sulfones ; Testosterone ; blood ; physiology

The diagnosis and treatment of erectile dysfunction has changed dramatically since the availability of safe and effective oral therapies. Unfortunately, not all men can be adequately treated in this way, and might require more invasive testing to diagnose and treat the specific cause of their dysfunction. This review looks at the tests and strategies available for men who cannot be treated by oral therapy alone.
3',5'-Cyclic-GMP Phosphodiesterases ; antagonists & inhibitors ; Administration, Oral ; Angiography ; Blood Flow Velocity ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Enzyme Inhibitors ; administration & dosage ; therapeutic use ; Erectile Dysfunction ; diagnosis ; drug therapy ; Humans ; Injections ; Male ; Penis ; blood supply ; diagnostic imaging ; Phosphodiesterase Inhibitors ; therapeutic use ; Vasodilator Agents ; therapeutic use

AIMTo investigate the effect of icariin on erectile function and the expression of nitric oxide synthase (NOS) isoforms in castrated rats.

METHODSThirty-two adult male Wistar rats were randomly divided into one sham-operated group (A) and three castrated groups (B, C and D). One week after surgery, rats were treated with normal saline (groups A and B) or oral icariin (1 mg/[kg.day] for group C and 5 mg/[kg.day] for group D) for 4 weeks. One week after treatment, the erectile function of the rats was assessed by measuring intracavernosal pressure (ICP) during electrostimulation of the cavernosal nerve. The serum testosterone (ST) levels, the percent of smooth muscle (PSM) in trabecular tissue, and the expression of mRNA and proteins of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and phosphodiesterase V (PDE5) in corpus cavernosum (CC) were also evaluated.

RESULTSICP, PSM, ST and the expression of nNOS, iNOS, eNOS and PDE5 were significantly decreased in group B compared with those in group A (P 0.01). However, ICP, PSM and the expression of nNOS and iNOS were increased in groups C and D compared with those in group B (P 0.05). Changes in ST and the expression of eNOS and PDE5 were not significant (P 0.05) in groups C and D compared with those in group B.

CONCLUSIONOral treatment with icariin ( 98.6 % purity) for 4 weeks potentially improves erectile function. This effect is correlated with an increase in PSM and the expression of certain NOS in the CC of castrated rats. These results suggest that icariin may have a therapeutic effect on erectile dysfunction.

3',5'-Cyclic-GMP Phosphodiesterases ; genetics ; metabolism ; Animals ; Blood Pressure ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Drugs, Chinese Herbal ; pharmacology ; Erectile Dysfunction ; drug therapy ; metabolism ; Flavonoids ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Male ; Muscle, Smooth ; drug effects ; physiology ; Nitric Oxide Synthase ; genetics ; metabolism ; Nitric Oxide Synthase Type I ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Orchiectomy ; Penile Erection ; drug effects ; Penis ; drug effects ; enzymology ; Pressure ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Testosterone ; blood