FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by GRAGASIN, F. S.
Right arrow Articles by ARCHER, S. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by GRAGASIN, F. S.
Right arrow Articles by ARCHER, S. L.
(The FASEB Journal. 2004;18:1382-1391.)
© 2004 FASEB

The neurovascular mechanism of clitoral erection: nitric oxide and cGMP-stimulated activation of BKCa channels

FERRANTE S. GRAGASIN, EVANGELOS D. MICHELAKIS, ANGIE HOGAN, ROHIT MOUDGIL, KYOKO HASHIMOTO, XICHEN WU, SANDRA BONNET, AL HAROMY and STEPHEN L. ARCHER1

Department of Medicine (Cardiology) and the Vascular Biology Group, University of Alberta, Edmonton, Canada

1Correspondence: Cardiology Division, Department of Medicine, University of Alberta, WMC 2C2.36, 8440 112th St., Edmonton, Alberta, Canada, T6G 2B7. E-mail: sarcher{at}cha.ab.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Female sexual function is under-studied, and mechanisms of clitoral engorgement-relaxation are incompletely understood. Penile erection results from nitric oxide (NO) -induced cyclic guanosine monophosphate (cGMP) accumulation. cGMP-dependent protein kinase (PKG) activates large-conductance, calcium-activated potassium channels (BKCa), thereby hyperpolarizing and relaxing vascular and trabecular smooth muscle cells, allowing engorgement. We hypothesize rat clitorises relax by a similar mechanism. Rat clitorises express components of the proposed pathway: neuronal and endothelial NO synthases, soluble guanylyl cyclase (sGC), type 5 phosphodiesterase (PDE-5), and BKCa channels. The NO donor diethylamine NONOate (DEANO), the PKG activator 8-pCPT-cGMP, and the PDE-5 inhibitor sildenafil, cause dose-dependent clitoral relaxation that is inhibited by antagonists of PKG (Rp-8-Br-cGMPS) or BKCa channels (iberiotoxin). Electrical field stimulation induces tetrodotoxin-sensitive NO release and relaxation that is inhibited by the Na+ channel blocker tetrodotoxin or sGC inhibitor 1H-(1,2,4)oxadiozolo(4,3-a)quinoxalin-1-one. Human BKCa channels, transferred to Chinese hamster ovary cells via an adenoviral vector, and endogenous rat clitoral smooth muscle K+ current are activated by this PKG-dependent mechanism. Laser confocal microscopy reveals protein expression of BKCa channels on clitoral smooth muscle cells; these cells exhibit BKCa channel activity that is activated by both DEANO and sildenafil. We conclude that neurovascular derived NO causes clitoral relaxation via a PKG-dependent activation of BKCa channels. The BKCa channel is an appealing target for drug therapy of female erectile dysfunction.—Gragasin, F. S., Michelakis, E. D., Hogan, A., Moudgil, R., Hashimoto, K., Wu, X., Bonnet, S., Haromy, A., Archer, S. L. The neurovascular mechanism of clitoral erection: nitric oxide and cGMP-stimulated activation of BKCa channels.


Key Words: laser capture microdissection • protein kinase G • electrical field stimulation • sildenafil • phosphodiesterase 5 • nitric oxide electrode


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SEXUAL FUNCTION IN FEMALES has not been widely studied and is often assumed to be different from that of males, with dysfunction resulting more from impaired desire than from impaired erectile function. More than 30% of women experience sexual dysfunction, and the prevalence increases with age, after menopause, and with a decline in circulating estrogen levels (1) . In males, penile erection is a neurovascular phenomenon in which sexual stimulation leads to local production of nitric oxide (NO) from nonadrenergic-noncholinergic (NANC) nerves and endothelial cells (2 3 4) . NO relaxes cavernous smooth muscle cells (SMC) and increases arterial inflow to the sinusoids, promoting penile tumescence. Penile erection results in large part from NO-induced activation of guanylyl cyclase, which causes accumulation of the second messenger cyclic guanosine monophosphate (cGMP). cGMP elicits relaxation through various mechanisms including hyperpolarization of arterial and cavernosal SMC. SMC hyperpolarization results from activation of cGMP-dependent protein kinase (PKG), which in turn phosphorylates and activates large-conductance, calcium-activated potassium channels (BKCa), a vasodilator pathway widely conserved among vascular beds and across species (5 , 6) . Disorders of this pathway at multiple levels (i.e., too little cGMP, too few BKCa channels) can result in erectile dysfunction (7) . Modern phosphodiesterase (PDE) -5 inhibitors, such as sildenafil, vardenafil, or tadalafil, enhance erectile function by slowing the rapid degradation of cGMP. These drugs preferentially target PDE-5, and this isoform has a relatively tissue-specific distribution, being most abundant in the penis, clitoris, and pulmonary circulation (8) .

Although the complete pathway of clitoral erection has not yet been elucidated, preliminary reports suggest that PDE-5 inhibitors may also be beneficial in women (9) . Upon sexual stimulation of the female, an increase in blood flow supplied by the dorsal clitoral and cavernosal clitoral arteries fills sinusoids contained in the corpora cavernosa (10 , 11) . As in males, the increase in clitoral blood supply is a result of arterial vasodilatation and smooth muscle relaxation in the corpus cavernosum, and this leads to increased clitoral size and tumescence (10 , 11) . Physical stimulation of the tumescent clitoris provokes pelvic floor muscle contraction, which triggers the female orgasm (12) . Indeed, failure to achieve clitoral tumescence may be an important factor in female sexual dysfunction (13) .

The current investigation evaluates two hypotheses: 1) NO and sildenafil cause clitoral relaxation through a PKG- and BKCa channel-dependent mechanism; 2) electrical field stimulation (EFS), a common experimental means of simulating sexual stimulation in erectile tissue, causes NO synthesis and induces relaxation of the rat clitoris via this same mechanism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
This study was approved by the Animal Health Care Committee of the University of Alberta, Edmonton.

Drugs
Diethylamine-NONOate (DEANO), NG-nitro-L-arginine methyl ester (L-NAME), 1H-(1,2,4)oxadiozolo(4,3-a)quinoxalin-1-one (ODQ), and iberiotoxin (IBTx) were purchased from Sigma-Aldrich (St. Louis, MO, USA); the PKG inhibitor 8-bromoguanosine-3',5'-cyclic monophosphorothioate-Rp isomer (Rp-8-Br-cGMPS) and activator 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMPS) were purchased from BIOLOG Life Science Institute (Bremen, Germany); sildenafil was a gift from Pfizer Pharmaceuticals (Sandwich, England). All drugs were dissolved in purified water except for ODQ, IBTx, and sildenafil, which were dissolved in dimethylsulfoxide (DMSO).

Tissue isolation
Clitoral relaxation was studied in rat clitoral strips under two conditions: preconstricted with the {alpha}-adrenoreceptor agonist phenylephrine or passively stretched to approximate resting clitoral dimensions. Clitoral tissue was obtained from female Sprague-Dawley rats (250–350 g). The rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.). The entire clitoris, including the urethra and vagina, were rapidly excised and placed in iced Krebs solution (pH 7.35 to 7.45, PO2 120 mm Hg, PCO2 40 mm Hg, 4°C). The rats were then killed by an overdose of sodium pentobarbital (additional 50 mg/kg i.p.). The vaginal wall and urethra were dissected free from the clitoral body. Clitoral strips 5 mm in length were obtained and mounted in a 4 mL organ bath for 45 min in Krebs solution at 37°C. Optimal tension, defined as the tension at which maximum constriction to phenylephrine (PE) occurred, was determined experimentally to be 500 mg (see Fig. 2a ). In the passive stretch protocol, resting tensions from 400 to 1200 mg were tested.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 2. Relaxation of the clitoris induced by the NO donor DEANO and the cGMP analog 8-pCPT-cGMPS. a) Optimal resting tension, judged by optimization of phenylephrine (PE) constriction in the rat clitoris, is 500 mg; n = 9. b, c) Mean data and representative traces show that DEANO and 8-pCPT-cGMPS cause dose-dependent relaxation that is attenuated by inhibiting PKG (Rp-8-Br-cGMPS) or BKCa channels (IBTx), n = 7 per group. Net relaxation to DEANO or 8-pCPT-cGMPS differs between groups (*P<0.05 vs. control). The effect of inhibitors was analyzed by dose (+P<0.05 Rp-cGMPS vs. control; #P<0.05 IBTx vs. control). At maximal doses, relaxation to DEANO persists despite the inhibitors.

Protocols
To determine the pathway of NO-induced relaxation, DEANO (10–9–10–6 mol/L), an NO donor (14 , 15) was used to stimulate relaxation after phenylephrine constriction under three conditions: control (no inhibitors), PKG inhibition (Rp-8-Br-cGMPS, 30 µmol/L) (16) , and BKCa inhibition (IBTx, 200 nmol/L) (17) . 8-pCPT-cGMP (10–8–10–6 mol/L), a cell-permeable cGMP analog (18) , was used to stimulate relaxation in the presence and absence of IBTx. Sildenafil (10–6–10–4 mol/L) was used to induce relaxation after phenylephrine constriction in the presence of vehicle, Rp-8-Br-cGMPS, or IBTx.

Relaxation in response to EFS, a method used to stimulate nerves and induce relaxation, was also assessed. EFS (pulse frequency 7.5 Hz, current amplitude 20 mA, pulse duration 200 µs) was applied for 5–80 s continuous stimulation using an AD Instruments Dual BioAmp/Stimulator (Mountain View, CA, USA). Experimentally, the maximal relaxation after PE constriction was seen with 60 s of EFS. To determine whether the pathway of neural relaxation occurs through an NO- and cGMP-dependent pathway, the following blockers were used in the presence of EFS: tetrodotoxin (TTX, 10–5 mol/L), a selective Na+ channel inhibitor to block nerve conduction; L-NAME (10–4 mol/L), a nonisoform-specific NO synthase (NOS) inhibitor; and ODQ (10–5 mol/L), a soluble guanylyl cyclase inhibitor. A 30 µm NO electrode (World Precision Instruments; Sarasota, FL, USA) was placed in the organ bath immediately adjacent the clitoris to detect EFS-induced NO production, as described previously (19) .

Immunohistochemistry
This technique was performed on freshly isolated rat clitorises as described previously (20) .

Quantitative real-time PCR (qRT-PCR)
qRT-PCR was performed as described using an ABI PRISM 7700 (Applied Biosystems; Foster City, CA, USA) and an Applied Biosystems Kit 4309169 (21 , 22) . mRNA expression was presented as 2{Delta}{Delta}Ct (i.e., abundance of the mRNA species of interest relative to the housekeeping gene ribosomal 18S mRNA and a calibrator sample; refs 21 , 22 ). Primers were constructed for {alpha}-smooth muscle actin (GenBank X06801), von Willebrands factor (vWF; GenBank AJ224673), eNOS (GenBank AJ249546), nNOS (GenBank NM_052799), sGC (GenBank NM_017090), PDE5(GenBank D89093), and BKCa (GenBank AF135265) mRNA. Total RNA was extracted from clitoral tissue with the Rneasy Mini Kit (Qiagen; Mississauga, Ontario, Canada). Sense, antisense, and Taqman probes for {alpha}-smooth muscle actin targeted nucleotides 573–592, 653–673, and 624–651, respectively. Sense, antisense, and Taqman probes for vWF targeted nucleotides 84–104, 145–164, and 107–132, respectively. Sense, antisense, and Taqman probes for eNOS targeted nucleotides 315–337, 385–367, and 340–360, respectively. Sense, antisense, and Taqman probes for nNOS targeted nucleotides 2618–2636, 2748–2729, and 2691–2713, respectively. Sense, antisense, and Taqman probes for sGC targeted nucleotides 1917–1936, 2033–2042, and 1966–1996, respectively. Sense, antisense, and Taqman probes for PDE-5 targeted 2340–2358, 2421–2440, and 2360–2382, respectively. Sense, antisense, and Taqman probes for BKCa targeted nucleotides 2891–2912, 3011–2988, and 2937–2965. A BLAST search confirmed the specificity of all probes for their targeted genes.

Laser capture microdissection (LCM)
The PixCell LCM (Arcturus Engineering; Mountain View, CA, USA) uses a microscope platform combined with a low-energy infrared laser to melt a plastic capture film onto selected structures on 5 µm-thick clitoral sections, allowing targeted removal of clitoral artery, nerve, and trabecula. The LCM sample was captured in a cap (see Fig. 6 ) and analyzed by qRT-PCR as described (23) .



View larger version (54K):
[in this window]
[in a new window]
  		Figure 6. Compartmental expression of eNOS, sGC, PDE5, and BKCa channels. LCM of clitoris shows the selective dissection of arteries, nerves, and trabeculae. qRT-PCR confirms relative purity of arteries, nerves, and trabeculae (i.e., arterial samples are rich in SM {alpha}-actin and vWF whereas nerves express little of either). sGC was most abundant in arteries and eNOS was most abundant in trabeculae, whereas mRNA for BKCa channels and PDE-5 was present in all structures. n = 5–12/group.

Adenoviral delivery of human BKCa gene
Replication-deficient, serotype 5 adenoviruses encoding green fluorescent protein (GFP) and BKCa gene (titers ~1.5x109 pfu/mL) were constructed using the Adeasy-1 system, as described (22 , 24 , 25) . The human BKCa cDNA construct was a generous gift from Bristol Myers Squibb. Preliminary restriction endonuclease map of plasmid (pbluescript-II KS+) carrying BKCa showed a potential Pac I site at 1900 bp downstream of the starting codon. Pac I restriction endonuclease is important for linearization of the adenoviral backbone before transfecting into human embryonic kidney (HEK) 293 cells. Therefore, to avoid potential Pac I digestion of the BKCa cDNA construct, a site-directed mutagenesis was performed. Briefly, an oligonucleotide carrying two mutated base pairs was used as an internal primer for the PCR from the original plasmid. The mutations changed the base pair configuration of the cDNA construct without altering the amino acid sequence of the nascent BKCa protein. The mutated BKCa was propagated with TA Cloning kit and subsequently ligated in pAdTrack-CMV vector. The resultant pAdTrack-CMV-BKCa construct was linearized with a PmeI restriction endonuclease digest, transformed together with an adenoviral plasmid (Adeasy-1) into bacterial BJ5183 cells, and plated on LB agar containing kanamycin. The selected colonies containing BKCa were isolated, amplified, purified and linearized (PacI endonuclease digest), then transfected into HEK 293 cells with liposome transfection (LipofectAmine reagent). Purification of the adenovirus was done by stepwise discontinuous CsCl gradient. Titration of the adenovirus carrying BKCa was done as described (22 , 24 , 25) . A 12 h incubation achieves ~90% infection of Chinese hamster ovary (CHO) cells after 48 h of growth.

Electrophysiology
Whole cell electrophysiology was performed on enzymatically dispersed clitoral SMCs or (for the BKCa expression studies) CHO cells using conventional patch whole-cell technique. The reporter GFP was used to select infected CHO cells for study. Cells were studied in voltage clamp configuration at a holding potential of –70 mV, then depolarized from –70 to +70 mV in 10 mV stepwise pulses of 200 ms duration. The cell capacitance was manually measured. Current density was expressed in whole cell currents for each cell divided by the cell capacitance..

Laser confocal microscopy
Enzymatically dispersed clitoral smooth muscle cells were imaged using an Axiovert 100M inverted confocal microscope (Carl Zeiss; North York, Ontario, Canada). Cells were stained with 4',6-diamidino-2-phenylindole (DAPI, a nuclear stain) and imaged by fluorescence for smooth muscle actin and BKCa channels using immunofluorescence, similar to the standard immunohistochemistry protocol mentioned above.

Statistics
Values are expressed as mean ± SE. Intergroup differences were assessed by repeated-measures or factorial ANOVA, as appropriate, with post hoc analysis using Fisher’s probable least significant differences test. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clitoral anatomy (Fig. 1)
The clitoris is composed of muscular trabeculae with potential vascular spaces that admit blood from arterioles during engorgement. Clitoral arteries, nerves, and trabecular sinusoids and muscle were visualized using conventional histological staining. There is an abundance of nerves. Although there is no fibrous tunica, there is a substantial amount of collagen and elastic tissue that limits clitoral size during engorgement.



View larger version (102K):
[in this window]
[in a new window]
  		Figure 1. Proposed mechanism of clitoral relaxation and clitoral histology. a) Masson’s Trichrome stain showing close relationship of nitrergic nerves and clitoral arteries (100x). b) Proposed mechanism of clitoral engorgement: NO causes relaxation by opening BKCa channels via a cGMP-/PKG-dependent mechanism in trabecular and vascular smooth muscle. c, d) Hematoxylin and eosin stain and elastic vanGiesen (EVG) stain, respectively, show arteries (A), nerves (N), and veins (V) as well as sinusoids in the rat clitoris (20x).

NO and cGMP cause relaxation in the rat clitoris through PKG-dependent activation of BKCa channels (Figs. 2 , 3)
Clitoral strips constricted optimally to phenylephrine at a resting tension of 500 mg. Clitoral contraction was characterized by phasic oscillations and some spontaneous relaxation. DEANO caused a rapid, dose-dependent relaxation of preconstricted clitoral strips that was significantly inhibited by IBTx and Rp-8-Br-cGMPS, up to DEANO doses of 10–7 mol/L. However, at the highest dose (10–6 mol/L), DEANO reduced PE tone by ~100% in all three treatment groups (control, IBTx, Rp-8-Br-cGMPS) (Fig. 2) . The cGMP analog 8-pCPT-cGMP caused complete clitoral relaxation that was blocked by IBTx (Fig. 2) . Like DEANO, the PDE-5 inhibitor sildenafil completely relaxed the clitoris and, at lower doses, this was blocked by Rp-8-Br-cGMPS and IBTx (10–6–10–5 mol/L) (Fig. 3) . However, at the highest dose, sildenafil continued to relax the clitoris despite the presence of Rp-8-Br-cGMPS or IBTx. These findings demonstrate the importance of cGMP-PKG-BKCa channel axis to NO-induced clitoral relaxation, particularly at physiological doses of NO.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 3. Sildenafil induces clitoral relaxation. a) Representative traces and b) mean data showing sildenafil induces dose-dependent relaxation after PE constriction, which is attenuated by IBTx and Rp-8-Br-cGMPS. n = 6/group. Net relaxation to sildenafil is inhibited by Rp-cGMPS and IBTx (*P<0.05 vs. control). Differences at each dose of sildenafil were analyzed (+P<0.05 Rp-cGMPS vs. control; #P<0.05 IBTx vs. control).

EFS causes relaxation in part via an NO- and cGMP-dependent mechanism (Fig. 4)
EFS was employed to study clitoral relaxation in response to endogenous NO synthesis. EFS reduced PE tone ~90% and caused synthesis of NO by the rat clitoris that was readily detectable using an NO electrode (Fig. 4) . The Na+ channel blocker TTX, used to block nerve conduction in the clitoris, almost completely inhibited EFS-induced NO synthesis (Fig. 4c ) and significantly reduced relaxation in clitorises stimulated both during active tension (phenylephrine, Fig. 4d ) and during passive tension (Fig. 4e ). L-NAME, an inhibitor of nNOS and eNOS isoforms, prevented EFS-induced relaxation and NO synthesis (Fig. 4c, d ). Since the magnitude of NO synthesis is similar in the L-NAME and TTX groups, it appears that nNOS in nitrergic nerves is the predominant source of NO during EFS. ODQ blocked clitoral relaxation due to EFS and, as expected, did not significantly attenuate NO production (Fig. 4c, d ), demonstrating that the downstream signaling molecule in EFS is cGMP.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 4. EFS causes NO synthesis and clitoral relaxation. a) Representative traces demonstrating clitoral relaxation in response to EFS in the presence of the NOS blocker L-NAME, soluble guanylyl cyclase inhibitor ODQ, and Na+ channel blocker TTX. b) Representative traces demonstrating EFS-induced NO synthesis, as detected by an NO microelectrode. c) L-NAME, ODQ, and TTX inhibit relaxation caused by EFS, n = 5 per group; *P < 0.05 vs. control. d) L-NAME and TTX attenuate NO production; ODQ does not affect NO synthesis, n = 5/group; *P < 0.05 vs. control. e) TTX inhibits clitoral relaxation to EFS in passively stretched clitoral strips. TTX (filled bars) has effect at low resting tensions (i.e., around the range of 500 mg) but did not inhibit relaxation at high resting tension (i.e., 800 mg), n = 5/group; *P < 0.05 vs. control (open bars).

Localization of expression of components of the NO/cGMP pathway using immunohistochemistry (Fig. 5) and laser capture microdissection (Fig. 6)
Immunohistochemistry reveals diffuse expression of eNOS. Na+ channels are expressed in arteries, nerves, and trabecular muscle whereas nNOS is expressed in nerves, trabecula, and within arteries, only in the endothelium. qRT-PCR on mRNA from whole rat clitorises confirms expression of all the key components of the pathway: eNOS, nNOS, sGC, PDE5, and BKCa channels (Fig. 5 , inset). To determine the relative expression of pathway components in various clitoral compartments, LCM was used to selectively dissect an artery, nerve, and trabecula from each clitoral specimen (Fig. 6) . qRT-PCR of LCM samples demonstrates that eNOS is most abundant in trabecula and sGC is most abundant in arteries (Fig. 6) . PDE-5 and BKCa channel mRNA are ubiquitously expressed. The accuracy of LCM sampling for the intended compartment is supported by the finding of much more abundant smooth muscle actin and vWF in putative arterial vs. nerve samples.



View larger version (106K):
[in this window]
[in a new window]
  		Figure 5. Immunohistochemistry and qRT-PCR of rat clitoris. a, b) Smooth muscle actin staining (brown) of clitoral arteries and trabecular SMCs, respectively (100x). c) Positive eNOS staining (brown) of clitoral trabecula and arteries (20x). d) BKCa channel staining (brown) in clitoral trabecula (20x). e) vWF staining (brown) positive in only the endothelium of clitoral blood vessels (20x). f–h) Na+ channels are expressed (brown) in clitoral arteries, nerves, and trabecular muscle, respectively (100x). i–k) Positive nNOS staining (brown) in artery and nerve trabecula, respectively (100x). Inset: Whole clitoral quantitative RT-PCR demonstrates the presence of BKCa channels, eNOS, nNOS, PDE5, soluble GC, SM actin, and vWF, where 40-Ct represents 40 (the upper limit of cycle time detection by the ABI Prism 7700) minus the cycle time (Ct) of when amplification of the mRNA of interest occurs (i.e., earlier amplification as a result of greater RNA quantity results in a lower Ct value). n = 2/group.

Electrophysiology (Figs. 7 , 8)
To determine the mechanism of interaction of NO with BKCa channels, we infected CHO cells with replication-deficient adenoviruses encoding the reporter green fluorescent protein (GFP) and BKCa channels. CHO cells do not functionally express BKCa channels and without infection have only a nonvoltage-gated, ohmic background current. CHO cells expressing GFP were used for patch-clamping experiments (Fig. 7) . DEANO increased BKCa channel activation and this was inhibited by Rp-8-Br-cGMPS, supporting the hypothesized mechanism of NO-induced clitoral engorgement (Fig. 1a ). To test this mechanism in clitoral SMCs, we freshly dispersed the SMCs, then confirmed their identity using immunofluorescence. This showed that the SMCs were positive for smooth muscle {alpha}-actin (green, Fig. 8a ) and expressed BKCa channels (red, Fig. 8a ). DEANO increased BKCa channel activation in these cells and this was inhibited by IBTx (Fig. 8b ). Sildenafil activated an iberiotoxin-sensitive BKCa current (Fig. 8c ).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 7. Infection of CHO cells with human BKCa channels yields a NO-activated, PKG-dependent K+ current. a) Representative traces showing that infection creates a voltage-gated current that is rapidly activating and noninactivating. b) Mean data showing DEANO increases K+ current in CHO cells expressing BKCa. This current is inhibited by the PKG inhibitor Rp-8-Br-cGMPS. a) CHO cells are selected for patch clamp study based on the expression of the reporter gene green fluorescent protein (25x).



View larger version (35K):
[in this window]
[in a new window]
  		Figure 8. Figure 8 . Clitoral smooth muscle cells exhibit BKCa channel activity activated by NO and sildenafil. a) Isolated clitoral SMCs visualized by confocal microscopy using differential interference contrast (DIC, gray), expressing smooth muscle {alpha}-actin (green), expressing BKCa channels (red), DAPI nuclear stain (blue), and the composite image showing colocalization (150x). b, c) Representative traces and mean data in clitoral SMC showing that DEANO and sildenafil, respectively, increases a K+ current that is sensitive to IBTx, demonstrating that these 2 agonists activate BKCa channels.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The goal of this study was to comprehensively establish the pathway of clitoral relaxation. The major finding is that clitoral relaxation ex vivo (and presumably engorgement in vivo) results from NO-mediated relaxation of trabecular and arterial SMCs by a mechanism involving cGMP-dependent, PKG-mediated activation of BKCa channels (Fig. 1a ). Inhibiting clitoral PKG, using Rp-8-Br-cGMPS, impairs NO/cGMP-mediated relaxation (Fig. 2) , consistent with the finding that male mice lacking cGMP-dependent kinase I have a reduced ability to reproduce and impaired relaxation of corpora cavernosa to NO and cGMP (26) . To our knowledge, this study offers the first measurement of clitoral NO production and the first description of clitoral SMC electrophysiology.

NO is a potent relaxer of clitoral tissue, reminiscent of its effects on penile tissue. We directly confirm, for the first time, that EFS induces measurable clitoral NO synthesis (Fig. 4b, d ). Moreover, the data indicate that the downstream targets of the neurally derived NO include PKG and BKCa channels. However, elevated cGMP levels, whether accomplished by enhancing synthesis (EFS), administering an NO donor (DEANO), or decreasing endogenous cGMP breakdown (sildenafil), cause PKG to activate BKCa channels (Fig. 7) . BKCa channel activation causes membrane hyperpolarization of SMCs and, as predicted, IBTx, a highly specific BKCa inhibitor, reduces relaxation to NO, cGMP, or sildenafil (Figs. 2 , 3) .

Figure 1 illustrates the proposed mechanism of relaxation used by NO and cGMP in the clitoris. The female rat has been validated as a model for the study of physiology and pharmacology with regard to sexual dysfunction relating to clitoral blood flow. Indeed, the use of the rat model is integral in the further research to understand the biology of female sexual function (27 , 28) .

Nitrergic nerves (4) and, more specifically, NO and cGMP (29) , have been shown to cause relaxation in rabbit clitoral corpus cavernosum. These meticulous studies showed that EFS induces nitrergic relaxation responses by a mechanism that is inhibited by TTX, ODQ, and L-NAME. These studies identified a central contribution of nitrergic nerves to rabbit clitoral relaxation to EFS but did not characterize the pathway distal to the level of sGC. The current work shows that engorgement (or its surrogate in this model, relaxation) results in large part from NO-initiated activation of BKCa channels by a PKG signaling cascade (Figs. 2 , 3) . EFS was effective in causing a reduction in clitoral tone both in phenylephrine-constricted and passively stretched clitoral preparations (Fig. 4c, e ). The resulting membrane hyperpolarization (Fig. 8) promotes relaxation by inhibiting the L-type Ca2+ channel, which is voltage sensitive (inhibited by hyperpolarization). Using the CHO expression system in which only BKCa channels were expressed (Fig. 7) , the effectiveness of NO and cGMP as channel activators was directly confirmed. However, in the clitoral strip, persistence of relaxation to NO, cGMP, and sildenafil at the highest doses, despite inhibition of PKG and BKCa channels, was observed. Perhaps the doses/tissue penetration of the inhibitors was inadequate. More probable is the explanation that at pharmacological doses, the mechanism of NO and cGMP is expanded to include less sensitive targets that lower cytosolic Ca2+ and promote relaxation [perhaps including other kinases, Ca2+ pumps and transporters in the sarcoplasmic reticulum (30) , and sarcolemma]. This phenomenon has been observed in arterial rings (6) , although the mechanism remains unclear.

Various PDE-5 inhibitors have been developed for human use (including sildenafil, vardenafil, and tadalafil) and they inhibit PDE-5 in cultured clitoral corpus cavernosal smooth muscle cells (31) , relax the corpus cavernosum of the rabbit clitoris (4 , 32) , and increase blood flow to the genitalia of the female dog (33) . Here, we show sildenafil causes relaxation of the rat clitoris, again in part, through a PKG- and BKCa-dependent mechanism. Sildenafil is a proven treatment of organic erectile dysfunction in males, and may improve sexual function in women on antidepressant medications, with spinal cord injury, or with sexual arousal disorder (9 , 34 , 35) . However, there is still controversy as to the efficacy/legitimacy of sildenafil in the treatment of female sexual dysfunction.

We demonstrate that the component enzymes and channels in the proposed pathway of clitoral relaxation (Na+ channels, nNOS, eNOS, sGC, BKCa) are present both as protein and as mRNA (Figs. 5 , 6) . Using LCM, we assessed the degree of compartmentalization of expression of pathway components. sGC, for example, is most abundant in clitoral arteries, eNOS in trabeculae, whereas PDE-5 and BKCa channels are ubiquitously expressed. The fact that none of the components were restricted to a single compartment demonstrates considerable redundancy in the pathway, which could promote preservation of an important reproductive pathway, consistent with the preserved fertility of eNOS and nNOS knockout mice. Moreover, the fact that all structures express each contributory component of the pathway probably reflects the fact that the full physiological response requires neural and endothelial NOS to achieve maximal relaxation of cavernosal muscle and vasodilation of clitoral arteries. These are separate pathways, which have an additive effect upon clitoral engorgement. Since PDE-5 is expressed in nerves as well as the vessels and trabeculae, PDE-5 may have neural effects, in addition to its better known effects on blood flow and smooth muscle tone. Although eNOS and PDE-5 have been identified in the human clitoris and vagina (31 , 36 , 37) , the distribution of expression in the structures (nerve, artery, and trabecula) has not previously been described.

Sexual dysfunction increases with age (38) , with erectile dysfunction being the main culprit in males but not necessarily in females (39) . However, organic (i.e., vascular) causes of sexual dysfunction in females may indeed play a role (13) given the increase in cardiovascular risk factors in the aging population, both male and female (40 41 42 43) . Hypertension in particular may impair clitoral erectile mechanisms at the arterial and neuronal levels (44) . Whether this sexual dysfunction associated with aging results from endothelial dysfunction (45) , an increase in clitoral collagen content (46) , or some combination of the two is not known. In the female, menopause may play a role, since it has been documented that a decrease in estrogen impairs clitoral engorgement in the rabbit (47) . Estrogen has relevance for the proposed NO-cGMP-K+ channel (including BKCa) mechanism (48 49 50) , because this sex hormone can increase eNOS protein expression (51 , 52) and directly activate BKCa channels (53) .

Although the incidence of erectile dysfunction increases with age, it is not known whether this results from decreased expression or impaired function of pathway components (particularly NOS and BKCa channels). In rats, the loss of male erectile function occurs with age and can be experimentally reversed by gene therapy with genes that enhance the production and survival of NO [e.g., superoxide dismutase, which would increase NO bioavailability (54) and eNOS (55 56 57) ]. Moreover, Christ et al. (7) demonstrated that hSlo cDNA, which encodes for the human smooth muscle BKCa channel, increases penile erection in the aged rat (7) . Since the serotype 5 adenovirus can reliably infect nondividing cells in vivo (25) , it will be possible to use the new vector reported in this paper to investigate BKCa channel replacement therapy in the female rat.

Extensive work characterizing the physiology of penile erection has resulted in successful clinical therapies, such as sildenafil. Based on this improved understanding of this pathway, one can propose and test strategies in treating female sexual dysfunction. An interesting future direction of study will be to examine the differential expression/activity of protein kinase G and BKCa channels with age and examine the effects of augmenting these targets using drugs or gene transfer strategies.


   ACKNOWLEDGMENTS
 
E.D.M. and S.L.A. are supported by the Canada Foundation for Innovation, the Alberta Heart and Stroke Foundation (H&SF), the Alberta Heritage Foundation for Medical Research (AHFMR), and the Canadian Institutes for Health Research (CIHR). S.L.A. is supported by NIH-RO1-HL071115.

Received for publication April 4, 2004. Accepted for publication May 17, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Berman, J. R., Berman, L. A., Werbin, T. J., Flaherty, E. E., Leahy, N. M., Goldstein, I. (1999) Clinical evaluation of female sexual function: effects of age and estrogen status on subjective and physiologic sexual responses. Int. J. Impot. Res. 11(Suppl. 1),S31-S38
  2. Kim, Y. C., Davies, M. G., Hagen, P. O., Carson, C. C., III (1994) Experimental evidence for endothelium dependent relaxation and neuronal nitric oxide in corpus cavernosum. Yonsei Med. J. 35,308-313[Medline]
  3. Ignarro, L. J., Bush, P. A., Buga, G. M., Wood, K. S., Fukuto, J. M., Rajfer, J. (1990) Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle. Biochem. Biophys. Res. Commun. 170,843-850[CrossRef][Medline]
  4. Cellek, S., Moncada, S. (1998) Nitrergic neurotransmission mediates the non-adrenergic non-cholinergic responses in the clitoral corpus cavernosum of the rabbit. Br. J. Pharmacol. 125,1627-1629[CrossRef][Medline]
  5. Robertson, B. E., Schubert, R., Hescheler, J., Nelson, M. (1993) cGMP-dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells. Am. J. Physiol. 265,C299-C303
  6. Archer, S. L., Huang, J. M., Hampl, V., Nelson, D. P., Shultz, P. J., Weir, E. K. (1994) Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 91,7583-7587[Abstract/Free Full Text]
  7. Christ, G. J., Rehman, J., Day, N., Salkoff, L., Valcic, M., Melman, A., Geliebter, J. (1998) Intracorporal injection of hSlo cDNA in rats produces physiologically relevant alterations in penile function. Am. J. Physiol. 275,H600-H608
  8. Wallis, R., Corbin, J., Francis, S., Ellis, P. (1999) Tissue distribution of phosphodiesterase families and the effects of sildenafil on tissue cyclic nucleotides, platelet function and the contractile responses of trabeculae carneae and aortic rings in vitro. Am. J. Cardiol. 83,3C-12C[Medline]
  9. Berman, J. R., Berman, L. A., Lin, H., Flaherty, E., Lahey, N., Goldstein, I., Cantey-Kiser, J. (2001) Effect of sildenafil on subjective and physiologic parameters of the female sexual response in women with sexual arousal disorder. J. Sex Marital Ther. 27,411-420[Medline]
  10. Levin, R. J. (1980) The physiology of sexual function in women. Clin. Obstet. Gynaecol. 7,213-252[Medline]
  11. Wagner, G. (1992) Aspects of genital physiology and pathology. Semin. Neurol. 12,87-97[Medline]
  12. Gillan, P., Brindley, G. S. (1979) Vaginal and pelvic floor responses to sexual stimulation. Psychophysiology 16,471-481[Medline]
  13. Park, K., Goldstein, I., Andry, C., Siroky, M. B., Krane, R. J., Azadzoi, K. M. (1997) Vasculogenic female sexual dysfunction: the hemodynamic basis for vaginal engorgement insufficiency and clitoral erectile insufficiency. Int. J. Impot. Res. 9,27-37[CrossRef][Medline]
  14. Morley, D., Keefer, L. K. (1993) Nitric oxide/nucleophile complexes: a unique class of nitric oxide-based vasodilators. J. Cardiovasc. Pharmacol. 22(Suppl. 7),S3-S9
  15. Hampl, V., Tristani-Firouzi, M., Hutsell, T. C., Archer, S. L. (1996) Nebulized nitric oxide/nucleophile adduct reduces chronic pulmonary hypertension. Cardiovasc. Res. 31,55-62[CrossRef][Medline]
  16. Nakazawa, M., Imai, S. (1994) Rp-8-Br-guanosine-3',5'-cyclic monophosphorothioate inhibits relaxation elicited by nitroglycerin in rabbit aorta. Eur. J. Pharmacol. 253,179-181[CrossRef][Medline]
  17. Candia, S., Garcia, M. L., Latorre, R. (1992) Mode of action of iberiotoxin, a potent blocker of the large conductance Ca2+-activated K+ channel. Biophys. J. 63,583-590[Medline]
  18. Wei, J. Y., Cohen, E. D., Genieser, H. G., Barnstable, C. J. (1998) Substituted cGMP analogs can act as selective agonists of the rod photoreceptor cGMP-gated cation channel. J. Mol. Neurosci. 10,53-64[Medline]
  19. Tristani-Firouzi, M., DeMaster, E. G., Quast, B. J., Nelson, D. P., Archer, S. L. (1998) Utility of a nitric oxide electrode for monitoring the administration of nitric oxide in biologic systems. J. Lab. Clin. Med. 131,281-285[CrossRef][Medline]
  20. Thebaud, B., Michelakis, E., Wu, X. C., Harry, G., Hashimoto, K., Archer, S. L. (2002) Sildenafil reverses O2 constriction of the rabbit ductus arteriosus by inhibiting type 5 phosphodiesterase and activating BK(Ca) channels. Pediatr. Res. 52,19-24[Medline]
  21. Michelakis, E. D., Hampl, V., Nsair, A., Wu, X., Harry, G., Haromy, A., Gurtu, R., Archer, S. L. (2002) Diversity in mitochondrial function explains differences in vascular oxygen sensing. Circ. Res. 90,1307-1315[Abstract/Free Full Text]
  22. Michelakis, E. D., Rebeyka, I., Wu, X., Nsair, A., Thebaud, B., Hashimoto, K., Dyck, J. R., Haromy, A., Harry, G., Barr, A., et al (2002) O2 sensing in the human ductus arteriosus: regulation of voltage-gated K+ channels in smooth muscle cells by a mitochondrial redox sensor. Circ. Res. 91,478-486[Abstract/Free Full Text]
  23. Archer, S. L., Gragasin, F. S., Wu, X., Wang, S., McMurtry, S., Kim, D. H., Platonov, M., Koshal, A., Hashimoto, K., Campbell, W. B., et al (2003) Endothelium-derived hyperpolarizing factor in human internal mammary artery is 11,12-epoxyeicosatrienoic acid and causes relaxation by activating smooth muscle BK(Ca) channels. Circulation 107,769-776[Abstract/Free Full Text]
  24. He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W., Vogelstein, B. (1998) A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA 95,2509-2514[Abstract/Free Full Text]
  25. Pozeg, Z. I., Michelakis, E. D., McMurtry, M. S., Thebaud, B., Wu, X. C., Dyck, J. R., Hashimoto, K., Wang, S., Moudgil, R., Harry, G., et al (2003) In vivo gene transfer of the O2-sensitive potassium channel Kv1.5 reduces pulmonary hypertension and restores hypoxic pulmonary vasoconstriction in chronically hypoxic rats. Circulation 107,2037-2044[Abstract/Free Full Text]
  26. Hedlund, P., Aszodi, A., Pfeifer, A., Alm, P., Hofmann, F., Ahmad, M., Fassler, R., Andersson, K. E. (2000) Erectile dysfunction in cyclic GMP-dependent kinase I-deficient mice. Proc. Natl. Acad. Sci. USA 97,2349-2354[Abstract/Free Full Text]
  27. Vachon, P., Simmerman, N., Zahran, A. R., Carrier, S. (2000) Increases in clitoral and vaginal blood flow following clitoral and pelvic plexus nerve stimulations in the female rat. Int. J. Impot. Res. 12,53-57[CrossRef][Medline]
  28. Munarriz, R., Kim, S. W., Kim, N. N., Traish, A., Goldstein, I. (2003) A review of the physiology and pharmacology of peripheral (vaginal and clitoral) female genital arousal in the animal model. J. Urol. 170,S40-S45[CrossRef][Medline]
  29. Park, J. K., Kim, J. U., Lee, S. O., Hwang, P. H., Yi, H. K., Kim, Y. G., Cho, K. W. (2002) Nitric oxide-cyclic GMP signaling pathway in the regulation of rabbit clitoral cavernosum tone. Exp. Biol. Med. (Maywood) 227,1022-1030[Abstract/Free Full Text]
  30. Twort, C. H., van Breemen, C. (1988) Cyclic guanosine monophosphate-enhanced sequestration of Ca2+ by sarcoplasmic reticulum in vascular smooth muscle. Circ. Res. 62,961-964[Abstract/Free Full Text]
  31. Park, K., Moreland, R. B., Goldstein, I., Atala, A., Traish, A. (1998) Sildenafil inhibits phosphodiesterase type 5 in human clitoral corpus cavernosum smooth muscle. Biochem. Biophys. Res. Commun. 249,612-617[CrossRef][Medline]
  32. Vemulapalli, S., Kurowski, S. (2000) Sildenafil relaxes rabbit clitoral corpus cavernosum. Life Sci. 67,23-29[CrossRef][Medline]
  33. Angulo, J., Cuevas, P., Cuevas, B., Bischoff, E., Saenz de Tejada, I. (2003) Vardenafil enhances clitoral and vaginal blood flow responses to pelvic nerve stimulation in female dogs. Int. J. Impot. Res. 15,137-141[CrossRef][Medline]
  34. Sipski, M. L., Rosen, R. C., Alexander, C. J., Hamer, R. M. (2000) Sildenafil effects on sexual and cardiovascular responses in women with spinal cord injury. Urology 55,812-815[CrossRef][Medline]
  35. Shen, W. W., Urosevich, Z., Clayton, D. O. (1999) Sildenafil in the treatment of female sexual dysfunction induced by selective serotonin reuptake inhibitors. J. Reprod. Med. 44,535-542[Medline]
  36. Burnett, A. L., Calvin, D. C., Silver, R. I., Peppas, D. S., Docimo, S. G. (1997) Immunohistochemical description of nitric oxide synthase isoforms in human clitoris. J. Urol. 158,75-78[CrossRef][Medline]
  37. D’Amati, G., di Gioia, C. R., Bologna, M., Giordano, D., Giorgi, M., Dolci, S., Jannini, E. A. (2002) Type 5 phosphodiesterase expression in the human vagina. Urology 60,191-195[CrossRef][Medline]
  38. Johannes, C. B., Araujo, A. B., Feldman, H. A., Derby, C. A., Kleinman, K. P., McKinlay, J. B. (2000) Incidence of erectile dysfunction in men 40 to 69 years old: longitudinal results from the Massachusetts male aging study. J. Urol. 163,460-463[CrossRef][Medline]
  39. Avis, N. E. (2000) Sexual function and aging in men and women: community and population-based studies. J. Gend. Specif. Med. 3,37-41
  40. Burchardt, T., Burchardt, M., Karden, J., Buttyan, R., Shabsigh, A., de la Taille, A., Ng, P. Y., Anastasiadis, A. G., Shabsigh, R. (2000) Reduction of endothelial and smooth muscle density in the corpora cavernosa of the streptozotocin induced diabetic rat. J. Urol. 164,1807-1811[CrossRef][Medline]
  41. Park, K., Ahn, K., Chang, J. S., Lee, S. E., Ryu, S. B., Park, Y. I. (2002) Diabetes induced alteration of clitoral hemodynamics and structure in the rabbit. J. Urol. 168,1269-1272[CrossRef][Medline]
  42. Seftel, A. D., Vaziri, N. D., Ni, Z., Razmjouei, K., Fogarty, J., Hampel, N., Polak, J., Wang, R. Z., Ferguson, K., Block, C., et al (1997) Advanced glycation end products in human penis: elevation in diabetic tissue, site of deposition, and possible effect through iNOS or eNOS. Urology 50,1016-1026[CrossRef][Medline]
  43. Park, K., Tarcan, T., Goldstein, I., Siroky, M. B., Krane, R. J., Azadzoi, K. M. (2000) Atherosclerosis-induced chronic arterial insufficiency causes clitoral cavernosal fibrosis in the rabbit. Int. J. Impot. Res. 12,111-116[CrossRef][Medline]
  44. Bechara, A. J., Cao, G., Casabe, A. R., Romano, S. V., Toblli, J. E. (2003) Morphological modifications in clitoris and vagina in spontaneously hypertensive rats. Int. J. Impot. Res. 15,166-172[CrossRef][Medline]
  45. Haas, C. A., Seftel, A. D., Razmjouei, K., Ganz, M. B., Hampel, N., Ferguson, K. (1998) Erectile dysfunction in aging: upregulation of endothelial nitric oxide synthase. Urology 51,516-522[CrossRef][Medline]
  46. Lin, J. S., Tsai, Y. S., Lin, Y. M., Lin, C. S., Chow, N. H. (2001) Age-associated changes in collagen content and its subtypes within rat corpora cavernosa with computerized histomorphometric analysis. Urology 57,837-842[CrossRef][Medline]
  47. Park, K., Ahn, K., Lee, S., Ryu, S., Park, Y., Azadzoi, K. M. (2001) Decreased circulating levels of estrogen alter vaginal and clitoral blood flow and structure in the rabbit. Int. J. Impot. Res. 13,116-124[CrossRef][Medline]
  48. Rosenfeld, C. R., Roy, T., Cox, B. E. (2002) Mechanisms modulating estrogen-induced uterine vasodilation. Vascul. Pharmacol. 38,115-125[CrossRef][Medline]
  49. Holdiman, A. J., Fergus, D. J., England, S. K. (2002) 17beta-Estradiol upregulates distinct maxi-K channel transcripts in mouse uterus. Mol. Cell. Endocrinol. 192,1-6[CrossRef][Medline]
  50. Node, K., Kitakaze, M., Kosaka, H., Minamino, T., Sato, H., Kuzuya, T., Hori, M. (1997) Roles of NO and Ca2+-activated K+ channels in coronary vasodilation induced by 17beta-estradiol in ischemic heart failure. FASEB J. 11,793-799[Abstract]
  51. MacRitchie, A. N., Jun, S. S., Chen, Z., German, Z., Yuhanna, I. S., Sherman, T. S., Shaul, P. W. (1997) Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium. Circ. Res. 81,355-362[Abstract/Free Full Text]
  52. Gragasin, F. S., Xu, Y., Arenas, I. A., Kainth, N., Davidge, S. T. (2003) Estrogen reduces angiotensin II-induced nitric oxide synthase and NAD(P)H oxidase expression in endothelial cells. Arterioscler. Thromb. Vasc. Biol. 23,38-44[Abstract/Free Full Text]
  53. Valverde, M. A., Rojas, P., Amigo, J., Cosmelli, D., Orio, P., Bahamonde, M. I., Mann, G. E., Vergara, C., Latorre, R. (1999) Acute activation of Maxi-K channels (hSlo) by estradiol binding to the beta subunit. Science 285,1929-1931[Abstract/Free Full Text]
  54. Bivalacqua, T. J., Armstrong, J. S., Biggerstaff, J., Abdel-Mageed, A. B., Kadowitz, P. J., Hellstrom, W. J., Champion, H. C. (2003) Gene transfer of extracellular SOD to the penis reduces O2–* and improves erectile function in aged rats. Am. J. Physiol. 284,H1408-H1421
  55. Bivalacqua, T. J., Champion, H. C., Mehta, Y. S., Abdel-Mageed, A. B., Sikka, S. C., Ignarro, L. J., Kadowitz, P. J., Hellstrom, W. J. (2000) Adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis improves age-related erectile dysfunction in the rat. Int. J. Impot. Res. 12(Suppl. 3),S8-17
  56. Bivalacqua, T. J., Usta, M. F., Champion, H. C., Adams, D., Namara, D. B., Abdel-Mageed, A. B., Kadowitz, P. J., Hellstrom, W. J. (2003) Gene transfer of endothelial nitric oxide synthase partially restores nitric oxide synthesis and erectile function in streptozotocin diabetic rats. J. Urol. 169,1911-1917[CrossRef][Medline]
  57. Champion, H. C., Bivalacqua, T. J., Hyman, A. L., Ignarro, L. J., Hellstrom, W. J., Kadowitz, P. J. (1999) Gene transfer of endothelial nitric oxide synthase to the penis augments erectile responses in the aged rat. Proc. Natl. Acad. Sci. USA 96,11648-11652[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by GRAGASIN, F. S.
Right arrow Articles by ARCHER, S. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by GRAGASIN, F. S.
Right arrow Articles by ARCHER, S. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS