1. Potentiation of penile tumescence by T-1032, a new potent and specific phosphodiesterase type V inhibitor, in dogs
T Noto, H Inoue, T Ikeo, K Kikkawa J Pharmacol Exp Ther. 2000 Sep;294(3):870-5.
We examined the mechanism underlying the potentiation of penile tumescence by methyl 2-(4-aminophenyl)-1, 2dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)3-isoquinoline carboxylate sulfate (T-1032), a new potent and selective phosphodiesterase type V inhibitor. In vivo, pelvic nerve stimulation induced a penile tumescence together with increase of total nitric oxide metabolite levels within the corpus cavernosa of anesthetized dogs. Intravenous (1-100 microg/kg) and intraduodenal (3, 30, 300 microg/kg) treatment with T-1032 dose dependently potentiated the tumescence. The potency of T-1032 was equivalent to that of sildenafil. T-1032 did not influence the intracavernous pressure when the pelvic nerve stimulation was absent. The potentiation of tumescence was more pronounced by intracavernous than i.v. injection. Intracavernous N(G)-nitro-L-arginine, a nitric-oxide synthase inhibitor, but not N(G)-nitro-D-arginine diminished the effects of T-1032 on the tumescence. Furthermore, i.v. T-1032 augmented the tumescence induced by sodium nitroprusside (SNP) but not by vasoactive intestinal polypeptide (VIP). In vitro, in isolated preparations of canine corpus cavernosum precontracted with phenylephrine, SNP (0. 01-100 microM) and VIP (0.01-1 microM) produced a dose-dependent relaxation accompanied by an increase in cGMP and cAMP levels, respectively. T-1032 augmented the relaxation induced by SNP but not by VIP. These data suggest that oral treatment with T-1032 has potential to improve erectile dysfunction through the inhibition of phosphodiesterase type V in the smooth muscles of corpus cavernosa.
2. Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide
N S Kwon, D J Stuehr, C F Nathan J Exp Med. 1991 Oct 1;174(4):761-7. doi: 10.1084/jem.174.4.761.
Macrophage-derived nitric oxide (NO) is cytostatic to tumor cells and microbial pathogens. We tested whether one molecular target for the cytostatic action of NO may be ribonucleotide reductase (RR), a rate-limiting enzyme in DNA synthesis. In a concentration-dependent manner, NO gas and lysates of activated macrophages that generated comparable amounts of NO led to the same degree of inhibition of partially purified RR from L1210 mouse lymphoma cells. Lysates from nonactivated macrophages, which do not produce NO, were noninhibitory. With lysates from activated macrophages, RR was protected by omitting L-arginine or by adding the NO synthase inhibitors diphenyleneiodonium, N omega-methyl-L-arginine, or N omega-amino-L-arginine. L-Arginine, but not D-arginine, abolished the protective effect of N omega-amino-L-arginine. The prototypic pharmacologic inhibitor of RR is hydroxyurea. Its structural resemblance to N omega-hydroxy-L-arginine, a reaction intermediate of NO synthase, prompted us to test if hydroxyurea can generate NO. In the presence of H2O2 and CuSO4, hydroxyurea produced NO2-/NO3-, aerobic reaction products of NO. Addition of morpholine blocked NO2-/NO3- generation from hydroxyurea and led to formation of nitrosomorpholine, as detected by gas chromatography/mass spectrometry. Thus, hydroxyurea can produce an NO-like, nitrosating rectant. L1210 cell DNA synthesis was inhibited completely by activated macrophages or by hydroxyurea, and was partially restored to the same degree in both settings by providing deoxyribonucleosides to bypass the block in RR. Thus, both NO gas and NO generated by activated macrophage lysates inhibit tumor cell RR. The RR inhibitor hydroxyurea can also generate an NO-like species. Similar, partial restoration of tumor cell DNA synthesis by deoxyribonucleosides in the presence of activated macrophages or hydroxyurea suggests that cytostasis by activated macrophages and by hydroxyurea has comparable mechanisms, including, but probably not limited to, inhibition of RR.
3. Analysis of serine proteases from marine sponges by 2-D zymography
Jeff G Wilkesman, Heinz C Schröder Electrophoresis. 2007 Feb;28(3):429-36. doi: 10.1002/elps.200600332.
Proteolytic activities isolated from the marine demosponges Geodia cydonium and Suberites domuncula were analyzed by 2-D zymography, a technique that combines IEF and zymography. After purification, a 200 kDa proteolytically active protein band was obtained from G. cydonium when analyzed in gelatin copolymerized 1-D zymograms. The enzymatic activity was quantified using alpha-N-benzoyl-D-arginine p-nitroanilide (BAPNA) as a substrate and corresponded to a serine protease. The protease activity was resistant to urea and SDS. DTT and 2-mercaptoethanol (2-ME) did not significantly change the protease activity, but induced a shift in molecular mass of the proteolytic band to lower M(r) values as detected by zymography. Under mild denaturing conditions, lower M(r) bands (<200 kDa) were identified in 1-D zymograms, suggesting that the protease is composed of subunits which retain the catalytic activity. After 2-D zymography, the protease from G. cydonium revealed a pI of 8.0 and an M(r) shift from 200 to 66 kDa. To contrast these results, a cytosolic sample from S. domuncula was analyzed. The proteolytic activity of this sponge after 2-D zymography corresponded to an M(r) of 40 kDa and a pI of 4.0. The biological function of both sponge proteases is not yet known. This study demonstrates that mild denaturing conditions required for IEF may alter the interpretation of the 2-D zymography, and care must be taken during sample preparation.
