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T F Lue

Publications and source records attributed to T F Lue.

At least 37 records · Page 2Linked to original sources
PubMed2001

Identification and regulation of human PDE5A gene promoter.

PDE5A gene encodes type 5 phosphodiesterase (PDE5), the principal cGMP-catalyzing enzyme in the penis and the primary target of sildenafil (Viagra). We have isolated a 3.7-kb DNA fragment that contains the human PDE5A gene promoter. The DNA fragment contains a sequence of 234 nucleotides at its 3' end that corresponds to most of the untranslated region of the PDE5A1 mRNA. The remaining 3.5-kb upstream flanking sequence contains no apparent TATA box but has several sequences that resemble binding sequences for transcription factors such as androgen receptor (AR), AP1, AP2, AP4, Sp1, MyoD, Myc, and CArG. In search of promoter activities, we used a luciferase reporter system to examine 10 DNA fragments that cover various regions of the 3.7-kb fragment. We found that a full basal promoter activity was confined to a 139-bp region that includes 78 bp of the PDE5A1-specific first exon. A lesser basal promoter activity was still detectable in a 94-bp fragment that contains the same 78-bp PDE5A1-specific exon plus 16 bp of upstream sequence. Either the 139-bp or the 94-bp promoter fragment responded minimally to cAMP or cGMP (2 mM) stimulation. Longer fragments that contain either a 308-bp 5' extension (from the 138-bp fragment) or a 156-bp 3' extension (all exon sequence) responded at higher levels to cAMP and cGMP stimulation. The 5' and 3' extensions cooperated with each other to provide the highest level of responses to cAMP and cGMP stimulation. DNase I footprint analysis identified four AP2- and two Sp1-binding sites in the 5' extension and four Sp1-binding sites in the 3' extensions. Cyclic AMP and cGMP had similar stimulatory effects on the PDE5A promoter.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2001

Regulation of human PDE5A2 intronic promoter by cAMP and cGMP: identification of a critical Sp1-binding site.

PDE5A gene encodes type 5 phosphodiesterase (PDE5), the principal cGMP-catalyzing enzyme in the penis and the primary target of sildenafil (Viagra). We have previously reported the isolation of three alternatively spliced PDE5A isoforms in humans. We also reported the identification of three corresponding alternative first exons and an intronic promoter in the human PDE5A gene. The intronic promoter is situated upstream from the PDE5A2-specific first exon but downstream from the PDE5A1- and A3-specific first exons. In the current study we showed that the intronic promoter could be upregulated by either cAMP or cGMP. In order to identify possible regulatory elements in the promoter, we created deletion and base-substitution mutants targeting one AP2- and four Sp1-binding sequences. Loss of function of these mutants to bind to the respective transcription factors was verified by DNase I footprint analysis, and changes in promoter function were analyzed with a luciferase reporter system. Mutation of the AP2-binding sequence and deletion of the 3'-most Sp1-binding site (within the exon) had little effects on the basal or the cyclic nucleotide-inducible promoter functions. Mutation of the 5'-most Sp1-binding site had much more severe effects on the basal and the cyclic nucleotide-inducible promoter functions. Mutation of a neighboring site that contains two overlapping Sp1-binding sequences completely nullified the basal and cyclic nucleotide-inducible promoter activities. Thus, the PDE5A2 intronic promoter depends on the overlapping Sp1-binding site for basal and cyclic nucleotide-inducible functions.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2001

Neurogenic erectile dysfunction.

Penile erection is a neurovascular event modulated by psyche and hormones. Erectile dysfunction (ED) has been classified as psychogenic, arteriogenic, neurogenic, endocrinologic, and cavernosal, based on the organs that are involved in penile erection. Among these types, neurogenic ED may be the most common, probably because a deficiency of neurotransmitters is the final common pathway in many diseases and conditions. This review discusses the physiology, pathophysiology, diagnosis, and treatment of erectile function and dysfunction, as well as strategies for future research.

Erectile Dysfunction↗
PubMed2001

Peyronie's disease.

Peyronie's disease remains one of the most perplexing diseases in urology. With continued basic research in wound healing and scarring, the understanding and management of this frustrating disease will improve. Initial treatment of Peyronie's disease should be conservative, with expectant therapy and medical management. Once the penile curvature and plaque have stabilized, patients with severe deformity can be offered surgery depending on their symptoms and complaints. Patient selection is the key to proper treatment. Less experienced surgeons should limit themselves to medical management or simple surgical management of the disease, including plication or Nesbit procedures. Penile-lengthening procedures or the placement of complicated penile prosthesis should be reserved for surgeons with familiarity and expertise in this type of reconstruction. Education on the pathogenesis and natural history of the disease will allow the patient and his partner to make an informed decision in regards to treatment options and expected outcomes.

Humans↗
PubMed2001

Traumatic arteriogenic erectile dysfunction: a rat model.

We developed a rat model of traumatic arteriogenic erectile dysfunction (ED) for the study of vasculogenic ED. Bilateral ligation of the internal iliac artery was performed on 30 three-month old male Sprague-Dawley rats as an experimental group. The control group consisted of 12 rats which underwent dissection of the internal iliac artery without ligation. Before their euthanization at 3 days, 7 days, and 1 month (10 rats in the experimental group and four rats in the control group at each time point), erectile function was assessed by electrostimulation of the cavernous nerves. Penile tissues were collected for nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining, trichrome staining, electron microscopy and RT-PCR for transforming growth factor beta (TGF-beta1), insulin like growth factor-I (IGF-I) and fibroblast growth factors (FGF) mRNA expression. Electrostimulation of the cavernous nerves revealed a highly significant declining of the intracavernous pressure after 3 and 7 days. No significant recovery of erectile function was noted at 1 month. Histology showed degeneration of the dorsal nerve fibers in all experimental rats. There was little decrease in the bulk of intracavernous smooth muscle in the experimental rats euthanazed 7 and 30 days. NADPH diaphorase staining revealed a significant decrease in nitric oxide synthase (NOS) containing nerve fibers in the dorsal and intracavernosal nerves in all rats in the experimental group. Electron microscopy showed a variety of changes such as collapse of sinusoids, increased cell debris, fibroblast and myofibroblast loss, intracellular deposition of fat and collagen and fatty degeneration. RT-PCR revealed up-regulation of TGF-beta1 after 3 days but not after 7 days or 1 month. There is no significant difference in IGF-I or FGF expression between the experimental and control group. Bilateral ligation of internal iliac arteries produces a reliable animal model for traumatic arteriogenic ED. Further studies are needed to investigate the molecular mechanism of ED in this model.

Animals↗
PubMed2001

Application of molecular biology to impotence research.

To encourage further application of molecular biology in impotence research, we have compiled a list of techniques that have been or can be used in such endeavors. While by no means complete or perfect, the list encompasses both some of the most commonly used (such as RT-PCR) and some of the most promising (such as gene chip) methods. All three levels of the gene expression hierarchy, namely, DNA, RNA, and protein, are represented in the discussion. Whenever possible, each technique is discussed with references relevant to impotence research. Interested readers therefore can trace the original or the most recent research protocols for more detailed information.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2001

The effect of adeno-associated virus mediated brain derived neurotrophic factor in an animal model of neurogenic impotence.

PURPOSE: We tested the hypothesis that transfecting penile tissue with brain derived neurotrophic factor may facilitate neural recovery and erectile capability after cavernous nerve injury. MATERIALS AND METHODS: Of the 34 Sprague-Dawley rats used 10 underwent sham operation and 24 underwent bilateral cavernous nerve freezing and intracavernous injection of adeno-associated virus-LacZ (12) or adeno-associated virus-brain derived neurotrophic factor (12). Erectile function was assessed by cavernous nerve electrostimulation at 4 and 8 weeks, and samples of penile tissue and the major pelvic ganglia were evaluated histologically. RESULTS: In the brain derived neurotrophic factor group mean maximal intracavernous pressure plus or minus standard deviation was significantly higher than in the LacZ group at 4 and 8 weeks (58.5 +/- 11.7 cm. water versus 28.4 +/- 5.5 and 61.3 +/- 12.5 versus 37.7 +/- 7.9, respectively). In addition, in the brain derived neurotrophic factor group reduced nicotinamide adenine dinucleotide phosphate diaphorase staining and neuronal nitric oxide synthase immunostaining revealed significantly more positive nerve fibers in the dorsal nerves and cavernous tissue than in the LacZ group at each time point and the percent of neuronal nitric oxide synthase positive neurons in the major pelvic ganglia was also significantly greater. Moreover, in the LacZ group most neurons showed a light staining pattern with irregular contours and numerous vacuoles in the cytoplasm. CONCLUSIONS: Intracavernous injection of adeno-associated virus-brain derived neurotrophic factor may prevent the degeneration of neuronal nitric oxide synthase containing neurons in the major pelvic ganglia and facilitate the regeneration of neuronal nitric oxide synthase containing nerve fibers in penile tissue, thus, enhancing the recovery of erectile function after bilateral cavernous nerve injury.

Animals↗
PubMed2001

The effect of simulated birth trauma and/or ovariectomy on rodent continence mechanism. Part I: functional and structural change.

PURPOSE: We examined the changes in the lower urinary tract after delivery, intravaginal ballooning and/or ovariectomy. MATERIALS AND METHODS: The study included 10 virgin and 48 primiparous pregnant rats. Cystometry and the stress/sneeze test were performed in virgin and postpartum rats shortly after delivery and at 8 weeks before sacrifice. Half of the delivered animals underwent intravaginal balloon dilation. Four weeks later half in each group underwent ovariectomy. The rats were subdivided into group 1--delivery, group 2--delivery plus balloon inflation, group 3--delivery plus ovariectomy and group 4--delivery plus balloon inflation plus ovariectomy. Tissues from the bladder, bladder neck, urethra and levator were collected, analyzed by electron microscopy, and immunostained for caveolin-1, caveolin-3 and neuronal nitric oxide synthase. RESULTS: Higher bladder capacity was detected in postpartum than in virgin rats. Urine leakage on stress/sneeze testing increased significantly in groups 2 and 4. Electron microscopy revealed a significant decrease in sarcolemma caveolae in the smooth muscle of the bladder and urethra in groups 2 to 4. In the bladder neck in group 3 caveolae were increased in smooth muscle. In groups 2 to 4 in the smooth muscle of the bladder and urethra caveolin-1 was significantly decreased. Caveolin-3 and neuronal nitric oxide synthase in striated muscle also significantly decreased in groups 2 to 4. CONCLUSIONS: These findings suggest that birth trauma simulated by ballooning and ovariectomy may contribute to stress urinary incontinence. The alteration in smooth muscle caveolae as well as the membrane protein caveolin may have a role in functional alterations caused by birth trauma and ovariectomy.

Animals↗
PubMed2001

Vascular endothelial growth factor promotes proliferation and migration of cavernous smooth muscle cells.

PURPOSE: We investigated whether cavernous smooth muscle cells secrete vascular endothelial growth factor (VEGF), how they respond to VEGF and how age affects their ability to secrete and respond to VEGF. MATERIALS AND METHODS: Corpus cavernous tissues were isolated from rats of various ages and grown as monolayer cell cultures. Smooth muscle identity was determined by immunofluorescence staining. Secreted VEGF was measured with a VEGF enzyme-linked immunosorbent assay kit. Cell proliferation was assayed with a cell titer kit. Cell migration was measured with Transwell chambers. VEGF receptor (VEGFR) messenger RNA and proteins were identified by reverse transcription-polymerase chain reaction and immunoblotting, respectively. RESULTS: Cavernous smooth muscle cells from young (age 1 week) and old (age 28 months) rats secreted the least amounts of VEGF and those from 16-week-old rats secreted the most. VEGF stimulated the growth of cavernous smooth muscle cells at an optimal concentration of 12.5 ng./ml. At this concentration cavernous smooth muscle cells from 11-week-old and 28-month-old rats showed the highest and lowest growth rates, respectively. The optimal VEGF concentration for cavernous smooth muscle cell migration was 10 ng./ml. At this concentration cells from 4-week-old and 28-month-old rats showed the highest and lowest migratory rates, respectively. The expression of VEGFR-1 appeared to parallel the VEGF regulated growth rates of these cells. VEGFR-2 was undetectable in cavernous smooth muscle cells. CONCLUSIONS: Cavernous smooth muscle cells from rats of different ages secreted different amounts of VEGF and showed different abilities to respond to VEGF. Cavernous smooth muscle cells responded to VEGF in terms of cell proliferation and migration. Cavernous smooth muscle cells from rats of different ages expressed different levels of VEGFR-1 but did not express VEGFR-2.

Aging↗
PubMed2001

Decreased trabecular smooth muscle and caveolin-1 expression in the penile tissue of aged rats.

PURPOSE: Because decreased trabecular smooth muscle content is reportedly associated with vasculogenic impotence in men, we performed a rodent study to investigate the effect of aging on trabecular smooth muscle content and caveolin-1 protein expression in penile smooth muscle cells. MATERIALS AND METHODS: In 6 young (age 3 months) and 6 old (age 24 months) rats erectile function was evaluated by cavernous nerve stimulation. At sacrifice penile tissue samples were collected for Western blot analysis, Masson's trichrome staining, caveolin-1 immunostaining and electron microscopy. The percent of smooth muscle in the trabecular tissue was assessed by computer assisted image analysis. RESULTS: In the aged rats mean intracavernous pressure plus or minus standard deviation was decreased (70 +/- 8.8 versus 107 +/- 12.3 cm. water) and the latency period was increased (7.8 +/- 1.2 versus 4.5 +/- 0.5 seconds) significantly compared to values in the young rats (p <0.001). The mean ratio of trabecular smooth muscle-to-connective tissue was also significantly altered in old versus young rats (27% +/- 2.9% versus 42.1% +/- 5.1%, p <0.001). Immunostaining for caveolin-1 was noted in each group in the sarcolemma of smooth muscle cells and endothelium of trabecular sinusoids but the staining pattern was less intense and the percent of smooth muscle positive for caveolin-1 was decreased in aged versus young rats (17.9% +/- 2.5% versus 27.5% +/- 3.6%, p <0.001). Moreover, young trabecular smooth muscle cells had more caveolae in the sarcolemma on electron microscopy and a higher expression of caveolin-1 protein on Western blot analysis. In contrast, higher endothelial nitric oxide synthase protein expression was noted in the penile tissue of old rats. CONCLUSIONS: In these aged rats the decreased ratio of trabecular smooth muscle-to-collagen and the reduced expression of caveolin-1 may contribute to erectile dysfunction.

Aging↗
PubMed2000

Effect of testosterone on the number of NADPH diaphorase-stained nerve fibers in the rat corpus cavernosum and dorsal nerve.

OBJECTIVES: To elucidate the effect of testosterone on penile nerve supply. METHODS: Three groups of 10 rats each were assessed; two groups were castrated and the third underwent a sham operation (control). After castration, one group received subcutaneous injection of testosterone while the others received sesame oil. At 8 weeks, the rats underwent a functional analysis. The evaluation included a subcutaneous injection with apomorphine to study centrally mediated erection, and cavernous nerve electrostimulation and papaverine injection to study peripherally mediated erection. At death, a penile midshaft specimen was taken for nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining. RESULTS: In the apomorphine study, castrated rats had no erections, but the erectile function of those receiving testosterone was restored to the level of the controls. The mean numbers of NADPH-diaphorase-stained nerve fibers in the copora cavernosa and both dorsal nerves of castrated rats, at 165.8 +/- 20.0 and 271.3 +/- 21.1, respectively, were significantly lower than those of the controls, at 271.7 +/- 14.6 and 471.2 +/- 27.6, respectively. Those of the testosterone replacement group, at 290.7 +/- 10.1 and 500.7 +/- 23.9, respectively, recovered to the control level. The intracavernosal pressure decreased significantly in the absence of testosterone, both after electrostimulation and intracavernosal papaverine injection, and recovered to the control level after testosterone replacement. CONCLUSIONS: Our results indicate that testosterone acts on the nervous system to mediate erection. When it is absent, there may be downregulation of both the production and activity of nitric oxide (NO), thereby decreasing the response to peripheral stimulation via the NO pathway. Testosterone replacement may upregulate NO activity to the control level.

Animals↗
PubMed2000

Identification of three alternative first exons and an intronic promoter of human PDE5A gene.

In the accompanying paper we present evidence for the existence of three PDE5A isoforms that differed only in the 5' end of the mRNAs. In this paper we present evidence that the three isoform-specific 5' ends were encoded by three alternative first exons that were arranged in the order of A1-A3-A2. Because the isoform-specific mRNAs could be transcribed from individual promoters, DNA fragments of the two intronic regions (A1-A3 and A3-A2) were tested for possible promoter activities. The intron between A1- and A3-specific exons did not exhibit any promoter activities even in smooth muscle cells that expressed the A3 isoform (see accompanying paper). In contrast, the intron between A3- and A2-specific exons had promoter activities in PDE5A2-expressing COS-7 and smooth muscle cells. This intronic promoter was bound by transcription factors AP-2 and Sp1, but not by AP-1, as shown by DNase I footprint analysis. However, the sequence bound by AP-2 (5'-GGGAAACGCTCGCGGGAGAGTTGG) is unusual in that it bears little resemblance to the consensus AP-2-binding sequence.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

Expression of three isoforms of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in human penile cavernosum.

Inhibition of cGMP-specific phosphodiesterase type V (PDE5) has been shown to improve penile erection in patients with erectile dysfunction. We report here the cloning of three PDE5 isoforms from human penile tissues. Two of the isoforms were identical to PDE5A1 and PDE5A2, respectively, which had been isolated from nonpenile tissues. The third isoform was novel and hence called PDE5A3. The deduced amino acid sequence of PDE5A3 was the same as the C-terminal 823-residue sequence of PDE5A1 and PDE5A2. While PDE5A1 and A2 isoforms were expressed in all tissues examined, the A3 isoform was confined to tissues with a smooth muscle or cardiac muscle component. When expressed in COS-7 cells, PDE5A1, A2, and A3 isoforms had similar cGMP-catalytic activities with K(m) of 6.2, 5.75, and 6.06 microM, respectively. Their cGMP-catalytic activities were inhibited by zaprinast with IC(50) values of 3.2 microM, 1.3 microM, and 1.6 microM, respectively, and by sildenafil with IC(50) of 28, 14, and 13 nM, respectively.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

The effect of pregnancy and delivery on the function and ultrastructure of the rat bladder and urethra.

OBJECTIVE: To examine the effect of pregnancy and delivery on the function and ultrastructure of the bladder and urethra in rats. Material and methods The study comprised six virgin and 18 pregnant rats; both groups underwent cystometry (at the 19th day of gestation, and 2 days and 6 weeks after parturition). Tissues from the bladder and urethra were collected for electron microscopy, western blotting and immunostaining for caveolin-1 and caveolin-3. RESULTS: The bladder capacity was greater and the modified leak-point pressures lower in pregnant and 2-day postpartum rats than in virgin and 6-week postpartum rats. The residual volume was significantly higher in the pregnant group. Electron microscopy showed more sarcolemmal caveolae in the smooth muscle cells of both the bladder and urethra of virgin rats than in the other groups. Lipid droplets and subsarcolemmal mitochondria accumulated in pregnant and 2-day postpartum rats. Caveolin-1 protein was detected in the cytoplasmic membrane of urethra and bladder smooth muscle cells. Caveolin-3 was detected in the membrane of striated muscle in the intrinsic sphincter. Western blotting showed increased caveolin-1 protein expression in the bladder and urethra of 2-day postpartum rats; in contrast, levels of caveolin-1 were lower in pregnant rats than in virgin and 6-week postpartum rats. CONCLUSION: s During pregnancy there was a significant decrease in sarcolemmal caveolae and caveolin-1 in the smooth muscle cells of the rat bladder and urethra. The changes in caveolae and the membrane protein caveolin may play a role in the functional changes associated with pregnancy and after delivery.

Animals↗
PubMed2000

Delayed testosterone replacement restores nitric oxide synthase-containing nerve fibres and the erectile response in rat penis.

OBJECTIVE: To elucidate the effect of testosterone on penile innervation. Materials and methods Three groups of six rats each were assessed; two groups (1 and 2) were castrated and the third (group 3) underwent a sham operation (control). Eight weeks after castration, group 2 received a subcutaneous injection with testosterone. At 8 weeks, the rats in group 1 and 3 underwent a final functional analysis while those in group 2 did so at 12 weeks. The evaluation included a subcutaneous injection with apomorphine to study centrally mediated erection, and cavernosal nerve electrostimulation and papaverine injection to study peripherally mediated erection. At death a penile mid-shaft specimen was taken for NADPH-diaphorase staining. RESULTS: In the apomorphine study, castration resulted in significantly fewer yawns and erections than in the control, and those in group 2 significantly better central erectile function than in the controls. The mean (SEM) number of nitric oxide synthase (NOS)-containing nerve fibres in the corpora cavernosa and both dorsal nerves of castrated rats, at 46.2 (9.1) and 203 (32.1), respectively, were significantly lower than in rats in group 2, at 84.1 (11.2) and 300.6 (17.1), and than in the controls, at 88.6 (10.9) and 306.3 (22.9), respectively. The intracavernosal pressure decreased significantly in the absence of testosterone, both after electrostimulation and intracavernosal papaverine injection. However, there was no difference between the control and group 2 rats in either the number of NOS-containing nerve fibres or in the peripheral erectile functional study. CONCLUSIONS: Testosterone acts on the nervous system to mediate erection; when it is absent there may be down-regulation of both the production and activity of NO, thereby decreasing the response to peripheral stimulation via the NO pathway. The restoration of erectile function seen in rats in group 2 supports this phenomenon. Delayed testosterone replacement has no detrimental effect on the restoration of the erectile mechanism after castration.

Animals↗
PubMed2000

Upregulation of L-plastin gene by testosterone in breast and prostate cancer cells: identification of three cooperative androgen receptor-binding sequences.

L-Plastin is normally a leukocyte-specific actin-binding protein; it is also expressed in the majority of human cancer cell lines that are derived from many types of solid tumors. We have previously reported the isolation of the L-plastin gene promoter, in which we identified several potential steroid receptor-binding sequences. We now obtained evidence that L-plastin gene expression was positively regulated by testosterone in androgen receptor (AR)-positive prostate and breast cancer cells. DNase I footprint analysis identified three AR-binding elements (ARE) located in a 545-bp region approximately 1.1 kb upstream from the transcription initiation site. However, each of these three AREs exhibited very little testosterone/AR-responsive enhancer activities toward a test promoter (of the thymidine kinase gene) when tested in MCF-7 breast cancer cells. Their testosterone/AR responsiveness became evident only when two or three of them were combined. In PC-3 prostate cancer cells, cooperation among L-plastin AREs was still evident although individually they had moderate levels of testosterone/AR responsiveness. Thus, the three L-plastin AREs, despite their imperfect sequences compared with the consensus ARE, could cooperate with each other to become a potent testosterone/AR-responsive unit, which was likely responsible for the inducibility of the L-plastin gene by testosterone.

Base Sequence↗