ORIGINAL RESEARCH—BASIC SCIENCE
ORIGINAL RESEARCH—BASIC SCIENCE: Heme Oxygenase vs. Nitric Oxide Synthase in Signaling Mediating Sildenafil Citrate Action

https://doi.org/10.1111/j.1743-6109.2007.00533.xGet rights and content

ABSTRACT

Introduction

Heme oxygenase (HO) enzyme catalyzes the rate limiting step in oxidative degradation of heme to biliverdin and carbon monoxide (CO). CO has been shown to share many properties with nitric oxide (NO), including activation of guanyl cyclase, signal transduction, and gene regulation.

Aim

To assess the signaling pathways mediating cavernous tissues response to sildenafil citrate intake experimentally.

Main Outcome Measures

In dissected cavernous tissues; detection of HO-1, HO-2 and nueronal nitric oxide synthase (nNOS) gene expressions by reverse transcriptase polymerase chain reaction (RT-PCR), HO enzyme activity assay, HO-1, HO-2 protein detection by Western blot, cyclic guanosine monophosphate (cGMP) tissue levels by enzyme linked immunosorbent assay (ELISA) and histopathology.

Methods

Two hundred forty Sprague-Dawley rats divided into five equal groups were investigated: group (Gr) 1, controls received regular diet; Gr 2, received sildenafil citrate 4 mg/kg orally; Gr 3, received the same dose of sildenafil added to HO inducer, diferuloylmethane; Gr 4, received sildenafil added to HO inhibitor, zinc protoporphyrin, and Gr 5, received sildenafil kg orally by gastric tube. Gr 3 received the same dose of sildenafil added to HO inducer, added to nitric oxide synthase inhibitor, L-Nitroarginine methylester. Twelve rats from each group were sacrificed by cervical dislocation successively after 1/2, 1, 2, and 3 hours from the intake.

Results

HO-2 gene expression was demonstrated in all groups. HO-1 was not expressed in controls, expressed in Gr 2, accentuated in Gr 3, and attenuated in Gr 4 and 5. These results were confirmed by Western blot. The nNOS was expressed in controls, increased in Gr 2 and 3, and decreased in Gr 4 and 5. HO enzyme activity and cGMP levels were significantly elevated in Gr 2, accentuated in Gr 3, and significantly decreased in Gr 4 and 5 compared to controls. Vasodilatations were observed in cavernous tissues of histopathologic sections of Gr 2 and increased in those of Gr 3.

Conclusion

Sildenafil citrate actions may be mediated by up-regulation of HO-1 gene expression. Abdel Aziz MT, El-Asmer MF, Mostafa T, Mostafa S, Atta H, Abdel Aziz Wassef M, Fouad H, Rashed L, Sabry D, and Mahfouz S. Heme oxygenase vs. nitric oxide synthase in signaling mediating sildenafil citrate action.

Introduction

Penile erection is produced by increased blood flow to the corpora cavernosa (CC) made by opening of penile resistance vessels (helicine arteries), relaxation of CC sinusoids, and occlusion of the venous outflow [1]. The erectile response depends on nitric oxide (NO), produced by nerves and by vascular endothelium [2]. NO activates soluble guanylate cyclase (sGC), producing cGMP that signals via different receptors, including ion channels, phosphodiesterase, and protein kinase. Cyclic guanosine monophosphate (cGMP) acts as a signaling mediator in vasodilatation and regulation of vascular tone, activates cGMP-dependent protein kinase that activates Ca2+/adenosine triphosphatase (ATPase) pump extruding Ca2+ into the endoplasmic reticulum, and activates Ca2+/K efflux pump extruding Ca2+ across the plasma membrane. Consequently, smooth muscles relax as a result of decreased intracellular Ca2+[3].

This knowledge has helped in the development of oral treatment for erectile dysfunction, the selective phosphodiesterase type 5 (PDE5) inhibitors [4]. Sildenafil citrate was the first introduced PDE5 inhibitor in 1998. Besides its proved erectogenic effect, it also showed different beneficial nonerectogenic actions in managing pulmonary hypertension, endometrial thickening, gastrointestinal disorders, Raynaud's phenomenon, etc. [5].

Heme oxygenase (HO) enzyme catalyzes the rate limiting step in oxidative degradation of heme to biliverdin and carbon monoxide (CO). Two isoforms of HO exist as products of distinct genes; HO-1 is an inducible isoform widely distributed in the mammalian tissues such as the liver, kidney, and reticuloendothelial tissues and other tissues, whereas HO-2 is constitutively expressed [6]. CO, a gaseous messenger similar to NO, has been shown to share many properties with NO, including activation of guanyl cyclase, signal transduction, and gene regulation [7, 8].

Many biological processes mediated by NO are manifested through activation or inhibition of metalloproteins such as guanylate cyclase [9]. Although HO in itself is not a heme containing protein, it forms a transitory heme protein complex when bound to its substrate that could be subjected to activation or inhibition by NO. Hedlund et al. [10] showed that the cholinergic nerve terminal within the human cavernous tissues contain nitric oxide synthase (NOS), HO-1, and HO-2, suggesting that they comprise a distinct population of parasympathetic cholinergic nerves. Qiao and Xin [11] also found that NOS and HO/CO system have a complementary role in penile erection. Shamloul and Wang [12] demonstrated increased intracavernosal pressure response in hypertensive rats after chronic hemin treatment.

This experimental study aimed to assess the signaling pathways mediating cavernous tissues response to sildenafil citrate intake.

Section snippets

Experimental Animals

Two hundred forty Sprague-Dawley male rats (average weight 150–200 gm), bred in the Experimental Medicine Unit according to Institutional Animal Care and Use Committee (IACUC) guidelines were used in the study after institutional review board approval. They were divided into five equal groups; all were housed under constant environmental conditions and were fed on standard laboratory rat chow and distilled water. Acclimatization was achieved in 7 days, after which proposed doses of tested

Results

Mean HO enzyme activity (pmol bilirubin/mg protein/minute) in the cavernous tissues of the investigated groups was higher in the sildenafil citrate-treated group after 1/2 hour, and reached its peak after 1 hour compared to the controls, with significant differences. HO enzyme activity decreased after 2 hours and 3 hours successively with nonsignificant difference from controls (Table 1, Figure 1). Mean cavernous tissues cGMP levels (pmol/mg protein) followed the same pattern of HO enzyme activity.

Discussion

The impact of sildenafil has stimulated academic, clinical, and industrial researchers to conduct experiments aimed at better understanding of the mechanism underlying male erectile function, hoping to develop better treatment modalities [25, 26]. PDE5 is the major cGMP hydrolyzing enzyme in the penile CC and is an important regulator of NO-mediated smooth muscle relaxation [4]. In the present work, we used a dose of sildenafil citrate of 4 mg/kg which is equivalent to 50-mg dose in men [13].

Category 1

  • (a)

    Conception and Design

  • M. Talaat Abdel Aziz, Mohamed Farid El-Asmer, Taymour Mostafa

  • (b)

    Acquisition of Data

  • Laila Rashed, Dina Sabry, Hazem Atta

  • (c)

    Analysis and Interpretation of Data

  • Samia Mostafa, M. Abdel Aziz Wassef, Hanan Fouad, Soheir Mahfouz

Category 2

  • (a)

    Drafting the Article

  • Samia Mostafa, M. Abdel Aziz Wassef, Hanan Fouad, Laila Rashed, Dina Sabry

  • (b)

    Revising It for Intellectual Content

  • M. Talaat Abdel Aziz, Mohamed Farid El-Asmer, Taymour Mostafa, Soheir Mahfouz, Hazem Atta

Category 3

  • (a)

    Final Approval of the Completed Article

  • M.

Acknowledgments

This work was awarded the International Society for Sexual Medicine (ISSM)/Pfizer Congress project awards/2005 and was presented in the 12th World Congress of the ISSM, Cairo, Egypt, September 2006.

References (46)

  • E.G. Valente et al.

    PDE 1-arginine and PDE inhibitors counteract fibrosis in the Peyronie's fibrotic plaque and related fibroblast cultures

    Nitric Oxide

    (2003)
  • L. Serfass et al.

    Effect of heme oxygenase inhibitors on soluble guanylate cyclase activity

    Arch Biochem Biophys

    (1998)
  • R. Vidavalur et al.

    Sildenafil induces angiogenic response in human coronary arteriolar endothelial cells through the expression of thioredoxin, hemeoxygenase and vascular endothelial growth factor

    Vascul Pharmacol

    (2006)
  • W.J. Hellstrom

    The molecular basis of erectile physiology: From bench to bedside

    J Androl

    (2002)
  • M. Craven et al.

    Modulation of spontaneous Ca2+ activated Cl-currents in the rabbit corpus cavernosum by the nitric oxide cGMP pathway

    J Physiol

    (2004)
  • A.D. Seftel

    Phosphodiesterase type 5 inhibitors: Molecular pharmacology and interactions with other phosphodiesterases

    Curr Pharm Des

    (2005)
  • B. Cremers et al.

    Non erectile dysfunction application of sildenafil

    Herz

    (2003)
  • M.D. Maines

    The heme oxygenase system: Past, present and future

    Antioxid Redox Signal

    (2004)
  • I.A. Sammut et al.

    Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of heme oxygenase-1

    Br J Pharmacol

    (1998)
  • M.T. Abdel-Aziz et al.

    Effects of nitric oxide synthase and heme oxygenase inducers and inhibitors on molecular signaling of erectile function

    Clin Biochem Nut (Japan)

    (2005)
  • L. Qiao et al.

    Advances in the research about penile erection related genes

    Zhonghua Nan Ke Xue

    (2002)
  • A.S. Tajuddin et al.

    Effect of 50% ethanolic extract of Syzygium aromaticum (L.) Merr. & Perry. (clove) on sexual behavior of normal male rats

    BMC Complement Altern Med

    (2004)
  • A.S. Tajuddin et al.

    Aphrodisiac activity of 50% ethanolic extracts of Myristica fragrans Houtt. (nutmeg) and Syzygium aromaticum (L.) Merr. & Perry. (clove) in male mice: A comparative study

    BMC Complement Altern Med

    (2003)
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