BACKGROUNDThere is currently considerable interest in the potential value of selective inhibitors of cyclic nucleotide phosphodiesterase 4 in the treatment of asthma. However, whether they influence eosinophilic airway inflammation-associated cough remains unclear. The objective of this study was to investigate the effects of selective phosphodiesterase 4 inhibitor SB207499 on cough response and airway inflammation in guinea pigs sensitized and challenged with ovalbumin.

METHODSForty sensitized guinea pigs were randomly divided into four groups: control (n = 10), challenge (n = 10), SB207499 (n = 10) and aminophylline (n = 10), then challenged with aerosol of 1% ovalbumin or saline. Two hours later, animals were intraperitoneally injected with either saline, 25 mg/kg of SB207499 or aminophylline. At the 24th hour, the injection was repeated with 2.5 mg/kg and 25 mg/kg SB207499 or aminophylline, then cough response to inhaled capsaicin and airway responsiveness to methacholine inducing a 150% of the peak airway pressure to the baseline (PC150) was measured. Finally, total cell number and differentials in bronchoalveolar lavage fluid were analysed.

RESULTSThe cough frequency per 3 minutes and PC150 in the challenge group were (22 +/- 4) times/3 minutes and (198 +/- 54) microg/ml, which were significantly different from (6 +/- 2) times/3 minutes and (691 +/- 81) microg/ml in the control group (P < 0.05, respectively). The injection of 25 mg/kg SB207499 significantly inhibited the increased cough response and airway hyperresponsiveness, the cough frequency and PC150 in guinea pigs were (13 +/- 2) times/3 minutes and (680 +/- 81) microg/ml (P < 0.05), which differed significantly from (18 +/- 2) times/3 minutes and (400 +/- 86) microg/ml after the administration of the same dose of aminophylline (P < 0.05). The inhibition of SB207499 on cough response was dose-dependent. Similarly, SB207499 decreased the total cell number and percentage of eosinophils in bronchoalveolar lavage fluid to (2.1 +/- 0.5) x 10(6)/ml and (20 +/- 5)% respectively, which were significantly different from (3.2 +/- 0.5) x 10(6)/ml and (29 +/- 5)% in the aminophylline group (P < 0.05, respectively) or (4.2 +/- 0.7) x 10(6)/ml and (35 +/- 4)% in the challenge group (P < 0.05, respectively).

CONCLUSIONPhosphodiesterase 4 inhibitor may be more useful than aminophylline for cough associated with eosinophilic airway inflammation via inhibiting airway inflammation and airway hyperresponsiveness.

3',5'-Cyclic-AMP Phosphodiesterases ; antagonists & inhibitors ; Animals ; Bronchial Hyperreactivity ; drug therapy ; Bronchoalveolar Lavage Fluid ; cytology ; Cough ; drug therapy ; Cyclic AMP ; biosynthesis ; Cyclic Nucleotide Phosphodiesterases, Type 4 ; Cyclohexanecarboxylic Acids ; therapeutic use ; Dose-Response Relationship, Drug ; Guinea Pigs ; Male ; Nitriles ; Ovalbumin ; immunology ; Phosphodiesterase Inhibitors ; therapeutic use

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

AIMTo investigate the expression of recombinant human phosphodiesterase 3A (HPDE3A) using baculovirus expression system in Tn cell line.

METHODSThe HPDE3A cDNA was recombined with baculovirus, and then the recombinant was transfected into Tn cell line. The expression of HPDE3A in Tn cell line was detected and identified by the RT-PCR, SDS-PAGE and Western blotting.

RESULTSThe recombinant HPDE3A protein was stably expressed in Tn cell line and detected by the distinct morphological changes of Tn cell, RT-PCR, SDS-PAGE and Western blotting using polyclonal antibody. The M(w) of the recombinant protein was about 120 kD.

CONCLUSIONRecombinant HPDE3A can be expressed in Tn cell line using the baculovirus expression system, and thus provided the basic material for studying its bioactivity and application in screening for HPDE3A inhibitor.

3',5'-Cyclic-AMP Phosphodiesterases ; genetics ; metabolism ; Animals ; Baculoviridae ; genetics ; Cell Line ; Cyclic Nucleotide Phosphodiesterases, Type 3 ; Electrophoresis, Polyacrylamide Gel ; Moths ; cytology ; enzymology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo evaluate whether mRNA levels of Pde4d and Alox5ap were associated with hypertensive stroke and hypertension in stroke-prone renovascular hypertensive rats (RHRSP) which could simulate human being's hypertensive cerebral stroke.

METHODSFive groups were established: normotensive group, gradient hypertensive groups I, II and III(with contractive pressure of 140-159 mmHg, 160-179 mmHg and 180-199 mmHg respectively) and spontaneous stroke group. RNA from leukocytes in peripheral blood of each rat underwent real time PCR after reversed.

RESULTSThe mRNA levels of Pde4d and Alox5ap of spontaneous stroke group were statistically higher than that of the other groups. Expression of Pde4d of hypertensive group I was a bit higher than that of normotensive group and hypertensive groups II and III; as for Alox5ap, there was no statistical difference between normotensive group and all gradient hypertensive groups.

CONCLUSIONAnimal experiments come to conclusions that over-expression of Pde4d and Alox5ap are associated with hypertensive stroke but not with hypertension. Therefore, the two genes confer the risk of hypertensive stroke independent of traditional risk factors. It is speculated that over-expression of Pde4d and Alox5ap can motivate onset of hypertensive cerebral stroke by participating in inflammation of arterial walls.

5-Lipoxygenase-Activating Proteins ; Animals ; Carrier Proteins ; genetics ; Cyclic Nucleotide Phosphodiesterases, Type 3 ; genetics ; Cyclic Nucleotide Phosphodiesterases, Type 4 ; Gene Expression Regulation ; Hypertension ; complications ; genetics ; Membrane Proteins ; genetics ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Stroke ; complications ; genetics

PURPOSE: Interleukin-1beta(IL-1beta) and Tumor necrosis factor-alpha(TNF-alpha) are multifunctional cytokines that may play important roles both in the normal development of central nervous system and in the response of brain to diverse forms of injury. IL-1beta and TNF-alpha have potent proinflammatory action and the potential to modulate cell growth. Cerebral hypoxia-ischemia selectively stimulates IL-1beta and TNF-alpha gene expression in brain regions susceptible to irreversible injury in perinatal rats. Pentoxifylline, a cAMP phosphodiesterase inhibitor, attenuates hypoxic-ischemic brain injury in immature rats and inhibits TNF-alpha expression at the transcription level. We hypothesize that pentoxifylline would attenuate the expression of IL-1beta and TNF-alpha mRNA gene expression on hypoxic-ischemic brain injury in immature rats. METHODS: To elicit focal hypoxic-ischemic brain injury, 7-d-old(P7) rats underwent right carotid artery ligation, followed by 3 hr of hypoxia(fractional concentration of inspired O2=0.08). In 3 rats, pentoxifylline(40mg/kg) was injected into the intraperitoneal cavity immediately before and after hypoxia. The other 4 rats were given PBS solutions. IL-1beta and TNF-alpha mRNA content were measured by reverse transcription followed by polymerase chain reaction amplification(RT-PCR) in the samples prepared from the lesioned and contralateral hemispheres killed 4 hr post-hypoxia. cDNA were amplified with primers specific for IL-1beta and TNF-alpha. and also amplified with GAPDH primers which served as an internal control. RESULTS: In control group, hypoxia-ischemia induced IL-1beta and TNF-alpha mRNA expression from the lesioned hemisphere in immature rat brain. In pentoxifylline treated group, IL-1beta and TNF-alpha mRNA expression were attenuated at 4 hr post hypoxia- ischemia. CONCLUSION: Preteatment with pentoxifylline decreased incidence and severity of hypoxic-ischemic injury in immature rat brain. Pentoxifylline attenuated the expression of IL-1beta and TNF-alpha gene on hypoxic-ischemic injury in immature rat brain. IL-1beta and TNF-alpha may play important roles in the response of the developing brain to acute hypoxic-ischemic injury.
3',5'-Cyclic-AMP Phosphodiesterases ; Animals ; Anoxia ; Brain Injuries* ; Brain* ; Carotid Arteries ; Central Nervous System ; Cytokines ; DNA, Complementary ; Gene Expression* ; Hypoxia-Ischemia, Brain ; Incidence ; Ischemia ; Ligation ; Necrosis ; Pentoxifylline* ; Polymerase Chain Reaction ; Rats* ; Reverse Transcription ; RNA, Messenger* ; Tumor Necrosis Factor-alpha*

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