Nitric Oxide in the Penis:Scientific Discoveries and Clinical Applications

Arthur L. (Bud) Burnett, M.D., M.B.A., F.A.C.S.Patrick C. Walsh Professor of Urology

The James Buchanan Brady Urological InstituteJohns Hopkins Medicine

Baltimore, Maryland

Disclosure Statement: In accordance with the ACCME policy on relevant financial disclosure, I disclose financial relationships with the following entities: American Medical Systems, Auxilium Inc, Coloplast, Endo Pharmaceuticals, National Institutes of Health, Pfizer Inc, Reflexonic LLC

Acknowledgments

Urology♦ Biljana Musicki, Trinity Bivalacqua, Thomas Chang

Neuroscience♦ Solomon Snyder, David Bredt, K. Joseph Hurt

Cardiology♦ Charles Lowenstein, Hunter Champion, David Kass

Hematology♦ James Casella, Samuel Charache, Lewis Hsu

Reproductive Biology♦ Barry Zirkin, Terry Brown

Overview

Multiple actions of nitric oxide in the penis♦Physiologic penile erection

♦Penile vascular health

♦Homeostasis and interaction with other signaling molecules

Therapeutic relevance♦Erectile dysfunction

♦Recurrent ischemic priapism

♦Penile fibrosis

A New Observation…

Leads to a New Question…

and to a New Discovery

Nitric Oxide: A Factor in ErectileTissue Relaxation

Direct application of nitric oxide relaxes isolated muscle strips from the corpus cavernosum similar to nerve stimulation

In vitro tissue relaxation effects are blocked by inhibitors of nitric oxide synthesis

“Neurotransmitter identity doubt” – proof needed that nitric oxide is released from neurons

1. Ignarro LJ et al. BBRC 170:843-50, 1990.

2. Rajfer J et al. N Engl J Med 326:90-4, 1992.

Nitric Oxide: A Physiologic Mediator of Penile Erection

Arthur L. Burnett, Charles J. Lowenstein, David S. Bredt,

Thomas S. K. Chang, Solomon H. Snyder*

Nitric oxide (NO) is a cytotoxic agent of macrophages, a messenger molecule of neurons, and a vasodilator produced by endothelial cells. NO synthase, the synthetic enzyme for NO, was localized to rat penile neurons innervating the corpora cavernosa and to neuronal plexuses in the adventitial layer of penile arteries. Small doses of NO synthase inhibitors abolished electrophysiologically induced penile erections. These results establish NO as a physiologic mediator of erectile function.

Reprint Series

17 July 1992, Volume 257, pp. 401-403 Science

Nitric Oxide Synthase is Localized to the Autonomic Innervation of the

Human Penis

Burnett AL, et al. J Urol 150:73-6, 1993.

Control of Trabecular Smooth Muscle Contractility

Clinical Therapeutics: Target Sites

Burnett AL. J Androl 2002; 23:S20-6.

A New Observation…

Leads to a New Question…

and to a New Discovery

Nitric Oxide Synthase Knockout Mice

Developed to investigate the involvement of nitric oxide in various biological functions1

Focus on nNOS, eNOS, double NOS mutant mice in sexual physiology research

Challenge: these mice preserve copulatory ability!2

- Why, if this regulatory pathway is essential?

- Do alternative mechanisms permit sexual

function?

1. Huang PL. Semin Perinatol 24, 87-90, 2004

2. Burnett AL, et al. J Androl 23, 92-97, 2002

Neuronal NOS Knockouts eNOS-dependent mechanisms are retained1

Lack nNOS (which encodes exon 2) but preserve nNOS splice variants2

nNOS β is expressed and is physiologically relevant3

1. Burnett AL, et al. Molec Med 2, 288-296 (1996)2. Gonzalez-Cadavid NF, et al. Biol Reprod 63, 704-714 (2000)3. Hurt KJ, et al. Proc Natl Acad Sci 103, 3440-3443 (2006)

Endothelial NOS Knockouts

Display supra-normal erections to electrophysiologic stimulation1

Display attenuated erections to pharmacologic stimulation2

1. Burnett AL, et al. J Androl 23, 92-97 (2002)

2. Hurt KJ, et al. PNAS 99, 4061-4066 (2002)

www.pnas.org/cgi/doi/10.1073/pnas.052712499 PNAS I March 19, 2002 I vol. 99 I no. 6 I 4061-4066

NO/eNOS Amplification System

Molecular Mechanisms that Underlie Penile Erection

Regulation of eNOS is the Key to Penile Vascular Health

Towards Better Penile Health:Hypothesis

Vasoactive Therapy

Erectile Tissue Relaxation

Intrapenile Blood Flow Stimulation

Activation of Endothelial NOS

Penile Vascular Repair and Restoration

A New Observation…

Leads to a New Question…

and to a New Discovery

Erection Excess in eNOS-/- Mice

Burnett AL, et al. J Androl 23:92-7, 2002

PDE5 Dysregulation In Penile Erectile Tissue: Mechanism Of Priapism

NOS3 -/- mice had enhanced erectile response to CNS. eNOS gene transfer to the NOS3-/- mouse penis resulted in

neurogenic-mediated erectile responses similar to WT mice via an elevation of PDE5A expression/activity.

Thus, properly regulated PDE5 function under physiologically relevant NO signaling preserves normal erection physiology.

Therefore, if penile PDE5 expression is dysregulated priapism occurs.

Champion HC et al PNAS 102:1661-66, 2005.

Biochemical Activity Measurements in Sickle Cell Mouse Penes

SS-/- mice show a priapic phenotype, and this is associated with reductions of both NOS and PDE5A activities

A Reversed Nitrate Tolerance Mechanism?

Mechanism of Priapism

Interim Summary:Hypothesis

Sickle Cell Disease PDE5 Inhibitor Therapy

eNOS

eNOS

PDE5

PDE5

Priapism Restore Normal Penile Vascular Homeostasis

PenileVasculature Endothelial-NO

Endothelial-NO

?ROS ?

pre-CNS post-CNS0

4

8

12 *n=7

WT+sildSSSS+sild

*

****

WT

X Labels

pre-CNSpost-CNS

WTY SEM0.600.95

0.50180.6037

WT+sildY SEM0.761.15

0.64390.9517

SSY SEM

5.209.67

1.9671.724

SS+sildY SEM2.644.33

0.73490.9434

Freq

uenc

y(e

rect

ions

/hr)

Effect of Chronic PDE5 Inhibitor Therapyon Erectile Responses in Wild Type

and Transgenic Sickle Cell Mice

WT Sickle Sickle + sildenafil0

5

10

15

20

25ICS

*n=6

**

AUC

Pos

t IC

S (c

m2 )

WT

3 Week Sildenafil Treatment

Hemi Sickle+

Sildenafil

WTWT + Sildenafil (100 mg/kg day)SickleSickle + Sildenafil (100 mg/kg day)

A New Observation…

Leads to a New Question…

and to a New Discovery

Role of NO and cGMP in Erections

PDE5 inhibitor

Sexualstimulation

Smoothmusclerelaxation

Erection

Corpus cavernosum

NO

--

NANC

cGMP=cyclic guanosine monophosphate. GTP=guanosine triphosphate. NANC=nonadrenergic, noncholinergic neurons. NO=nitric oxide. PDE5=phosphodiesterase type 5.

I

Sustained phosphorylation of nNOS-S1412 after electrical stimulation of rat MPG.

Intracavernous pressure increases with intrapenile injection of forskolin in wild-type but not nNOS-/-mice or wild-type after L-NAME pretreatment.

PNAS 109(2012), 16624-9

Integrative Model of nNOS and eNOS Regulation of Initiation and Maintenance of Erectile Function

NOS Roles in Penile Biology

Function Isoform

Erection Mediation

Initiator nNOS α/β, eNOS

Facilitator P-nNOS, P-eNOS

Erectile Tissue Preservation

Vasculoprotector P-eNOS

Anti-fibrosis agent iNOS

Homeostasis

Biochemical Modulator eNOS

“Gladly I think of the dayswhen all my members were

limber, all except one

Those days are certainly gone, now all my members are stiff,

all except one”

- Goethe