Homocysteine and copper interact to promote type 5 phosphodiesterase expression in rabbit cavernosal smooth muscle cells.
OBJECTIVE
To study the effects of homocysteine and copper on type 5 phosphodiesterase (PDE5) expression in cavernosal vascular smooth muscle cells (CVSMCs) and to investigate superoxide (O(2)(.-)) derived from nicotinamide adenine dinucleotide phosphate oxidase as homocysteine and copper generate O(2)(.-), and O(2)(.-) upregulates PDE5 expression.


METHODS
CVSMCs derived from rabbit penis were incubated with homocysteine or copper chloride with or without superoxide dismutase (SOD), catalase, sildenafil citrate, or apocynin (nicotinamide adenine dinucleotide phosphate inhibitor) for 16 h. The expression of PDE5 and of glyceraldehyde-3-phosphate dehydrogenase (internal standard) was assessed using Western blot analysis. In parallel, O(2)(.-) was measured spectrophotometrically.


RESULTS
CuCl(2) alone (up to 10 micromol/L) and homocysteine alone (up to 100 micromol/L) had no effect on O(2)(.-) formation in CVSMCs compared to controls. In combination, however, homocysteine and CuCl(2) markedly increased O(2)(.-) formation, an effect blocked by SOD, catalase, apocynin, and sildenafil (1 micromol/L) when co-incubated over the same time course. PDE5 expression was also significantly increased in CVSMCs incubated with homocysteine and CuCl(2), compared to controls. This effect was also negated by 16-h co-incubation with SOD, catalase, apocynin and sildenafil.


CONCLUSIONS
This represents a novel pathogenic mechanism underlying ED, and indicates that the therapeutic actions of prolonged sildenafil use are mediated in part through inhibition of this pathway.
Hotston
Matthew
M
Department of Urology, University of Bristol, 13 Freeland Place, Bristol BS8 4NP, UK. matthotston@hotmail.com
Jeremy
Jamie Y
JY
Bloor
Jonathon
J
Greaves
Nick S
NS
Persad
Raj
R
Angelini
Gianni
G
Shukla
Nilima
N
D000818:Animals; D015153:Blotting, Western; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000096:biosynthesis*; D003627:Data Interpretation, Statistical; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006710:Homocysteine / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors; D010396:Penicillamine / Q000494:pharmacology; D010413:Penis / Q000187:drug effects / Q000201:enzymology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D000068677:Sildenafil Citrate; D013450:Sulfones / Q000494:pharmacology
D002614:Chelating Agents; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D017382:Reactive Oxygen Species; D013450:Sulfones; D006710:Homocysteine; D003300:Copper; D000068677:Sildenafil Citrate; D019255:NADPH Oxidases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D010396:Penicillamine