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Biochemical Pharmacology (v.74, #4)

Editorial Advisory Board (pp. iii).

Ins and outs of dietary phytochemicals in cancer chemoprevention by Gian Luigi Russo (pp. 533-544).

A voluminous number of evidence suggests that an increased consumption of fruit and vegetables is a relatively easy and practical strategy to reduce significantly the incidence of chronic diseases, such as cancer, cardiovascular diseases and other aging-related pathologies. This review will critically discuss the applications of chemical and dietary chemoprevention, intending the protecting effects against cancer of chemically synthesized molecules, or phytochemicals present in the regular diet. The length of chemopreventive treatments requires the administration of low doses of chemopreventive agents, to avoid toxic side effects. This poses the question, here discussed, of the bioavailability of these compounds, usually very modest. Another key issue is whether purified phytochemicals have the same protective effects, as do the whole food or mixture of foods in which these compounds are present. These aspects will be analysed at the light of the “antioxidant hypothesis” in cancer prevention and the “combination chemoprevention”, both referring to the pleiotropic and synergistic effects of compounds present in the diet. Single molecules may evolve in perfect chemopreventive agents, as in the case of tamoxifen, or generate ambiguity. Resveratrol and quercetin represent two paradoxes, discussed here.

Keywords: Phytochemicals; Polyphenols; Antioxidant; Chemoprevention; Resveratrol; Quercetin; Diet

The effect of the lipophilic cation lucigenin on mitochondria depends on the site of its reduction by Alexey G. Kruglov; Vera V. Teplova; Nils-Erik L. Saris (pp. 545-556).

The role of NAD(P)H-dependent oxidoreductases of the outer mitochondrial membrane (OMM) in the activation of lipophilic cationic dyes is poorly understood. In the present study we compared the rates of production of reactive oxygen species (ROS) and mitochondriotoxic effects of the redox-cycling lipophilic cationic dye lucigenin upon its activation by the respiratory chain and NAD(P)H-dependent oxidoreductases of the OMM. We found that, only in the presence of external NADH and NADPH, which are unable to penetrate the inner membrane, lucigenin stimulated a massive superoxide production and a fast permeabilization of mitochondrial membranes. The permeabilization was biphasic. The first, cyclosporin A-insensitive and Ca²⁺-independent phase was characterized by increased permeability of the inner mitochondrial membrane to solutes with molecular masses of ≤200Da. The second phase was sensitive to the antagonists of the permeability transition pore (mPTP) and was characterized by permeability similar to that of mPTP (≤1500Da). A massive cytochrome c release was observed even at the first phase of permeability when the second phase was inhibited by mPTP antagonists. Whatever the site of lucigenin activation, antioxidants and scavengers of ROS that strongly decrease the ROS level were unable to delay or prevent the permeabilization of membranes, which casts doubt on the involvement of ROS in the regulation of permeability by redox-cycling lipophilic cations. Our results strongly support the idea that the NAD(P)H-dependent reductases of xenobiotics of the OMM can mediate the toxicity of cationic dyes.

Keywords: Abbreviations; Δ; Ψ; _m; inner membrane transmembrane potential; BA; bongkrekic acid; BHT; 2,6-di-; tert; -butyl-4-methylphenol; CsA; cyclosporin A; DBA; dimethylbiacridene; FCCP; carbonyl cyanide; p; -trifluoromethoxyphenylhydrazone; IMM; inner mitochondrial membrane; MCLA; 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazine-3-one; MDCL; MCLA-derived chemiluminescence; mPTP; mitochondrial permeability transition pore; OMM; outer mitochondrial membrane; PEG; polyethylene glycol; PMC; 2,2,5,7,8-pentamethyl-6-chromanol; RLM; rat liver mitochondria; ROS; reactive oxygen species; SOD; superoxide dismutase; TEMPO; 4-hydroxy-2,2,6,6-tetramethylpiperidine-; N; -oxyl; TPP; ⁺; tetraphenylphosphoniumCationic dyes; Permeability transition pore; Outer mitochondrial membrane; NAD(P)H-dependent oxidoreductases; Xenobiotics; Reactive oxygen species

Apoptosis induction of 2′-hydroxycinnamaldehyde as a proteasome inhibitor is associated with ER stress and mitochondrial perturbation in cancer cells by Su Hyung Hong; Jina Kim; Jung-Min Kim; So-Young Lee; Dae-Seop Shin; Kwang-Hee Son; Dong Cho Han; Young Kwan Sung; Byoung-Mog Kwon (pp. 557-565).

2′-Hydroxycinnamaldehyde (HCA), isolated from the stem bark of Cinnamomum cassia, and 2′-benzoyloxycinnamaldehyde (BCA), one of HCA derivatives, have antiproliferative activities on several human cancer cell lines. Our previous study suggested that reactive oxygen species (ROS) and caspase-3 are the major regulators of HCA-induced apoptosis. In the present study, we demonstrated a novel molecular target using in vitro pull-down assay by biotin-labeled HCA (biotin-HCA) in SW620 cells. We analyzed 11 differential spots of 2-dimensional gel prepared with pull-downed proteins by biotin-HCA. Among them, five spots were identified as proteasome subunits. An in vitro 26S proteasome function assay using specific fluorogenic substrates showed that HCA potently inhibits L3-like activity of the proteasome. In addition, HCA showed inhibitory action against chymotrypsin-like, trypsin-like, and PGPH-like activities. DNA microarray showed that HCA induced heat shock family and ER stress-responsive genes, which reflects the accumulation of misfolded proteins by proteasome inhibition. On western blot analysis, it was confirmed that HCA induces glucose-regulated protein, 78kDa (GRP78) and some representative endoplasmic reticulum (ER) stress-responsive proteins. Furthermore, HCA treatment decreased mitochondrial membrane potential. The effect of HCA on cytochrome c and Bax translocation between cytosol and mitochondrial membrane was clarified using western blot analysis. These results suggest that HCA-induced apoptosis is associated with the inhibition of the proteasome activity that leads in turn to the increase of ER stress and mitochondrial perturbation.

Keywords: Abbreviations; HCA; 2′-hydroxycinnamaldehyde; BCA; 2′-benzoyl-oxycinnamaldehyde; ROS; reactive oxygen species; MCA; 4-methyl-coumaryl-7-amide; HMOX1; heme oxygenase 1; HERPUD1; homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1; GADD153/CHOP; growth arrest and DNA damage-inducible gene/C/EBP homologous protein; GRP78; glucose-regulated protein, 78; kDa2′-hydroxycinnamaldehyde; Apoptosis; Proteasome; Endoplasmic reticulum; Mitochondria

Design and cellular kinetics of dansyl-labeled CADA derivatives with anti-HIV and CD4 receptor down-modulating activity by Kurt Vermeire; Andrea Lisco; Jean-Charles Grivel; Emily Scarbrough; Kaka Dey; Noah Duffy; Leonid Margolis; Thomas W. Bell; Dominique Schols (pp. 566-578).

A new class of anti-retrovirals, cyclotriazadisulfonamide (CADA) and its derivatives, specifically down-regulate CD4, the main receptor of HIV, and prevent HIV infection in vitro. In this work, several CADA derivatives, chemically labeled with a fluorescent dansyl group, were evaluated for their biological features and cellular uptake kinetics. We identified a derivative KKD-016 with antiviral and CD4 down-modulating capabilities similar to those of the parental compound CADA. By using flow cytometry, we demonstrated that the dose-dependent cellular uptake of this derivative correlated with CD4 down-modulation. The uptake and activity of the dansyl-labeled compounds were not dependent on the level of expression of CD4 at the cell surface. Removal of the CADA compounds from the cell culture medium resulted in their release from the cells followed by a complete restoration of CD4 expression. The inability of several fluorescent CADA derivatives to down-modulate CD4 was not associated with their lower cellular uptake and was not reversed by facilitating their cell penetration by a surfactant. These results prove the successful integration of the dansyl fluorophore into the chemical structure of a CD4 down-modulating anti-HIV compound, and show the feasibility of tracking a receptor and its down-modulator simultaneously. These fluorescent CADA analogs with reversible CD4 down-regulating potency can now be applied in further studies on receptor modulation, and in the exploration of their potentials as preventive and therapeutic anti-HIV drugs.

Keywords: Abbreviations; HIV; human immunodeficiency virus; CADA; 9-benzyl-3methylene-1,5-di-; p; -toluenesulfonyl-1,5,9-triazacyclododecane (cyclotriazadisulfonamide); Dansyl; 5-dimethylamino-1-naphtalenesulfonyl; Tosyl; toluenesulfonyl; MFI; median fluorescence intensity; DMSO; dimethylsulfoxide; SI; selectivity indexCADA; Dansyl; Anti-HIV; CD4 receptor; Reversible Down-modulation; Cellular kinetics

Methylglyoxal suppresses TNF-α-induced NF-κB activation by inhibiting NF-κB DNA-binding by Mathias Laga; Anneleen Cottyn; Franky Van Herreweghe; Wim Vanden Berghe; Guy Haegeman; Patrick Van Oostveldt; Joël Vandekerckhove; Katia Vancompernolle (pp. 579-589).

Methylglyoxal is a cytotoxic metabolite that is produced in vivo mainly from glycolysis. Increased production of methylglyoxal can be induced by tumor necrosis factor and occurs in a number of pathological conditions, including diabetes and neurodegenerative disorders. Methylglyoxal is highly reactive and can modify proteins, which results in the formation of advanced glycation end products. Yet, we, and others, have recently proposed a role for methylglyoxal as a signaling molecule. In this study, we show that methylglyoxal inhibits TNF-induced NF-κB activation and NF-κB-dependent reporter gene expression by inhibiting the DNA binding capacity of NF-κB p65. Methylglyoxal slightly delayed, but did not inhibit, TNF-induced degradation of IκBα and strongly inhibited TNF-induced NF-κB-dependent re-synthesis of IκBα. The TNF-induced nuclear translocation of NF-κB p65 was also delayed, but not inhibited, in the presence of methylglyoxal. TNF-induced phosphorylation of p65 was not affected by methylglyoxal. We show that the conserved Cys 38 residue, which is located in the DNA binding loop of NF-κB p65 and responsible for the redox regulation of the transcription factor, is involved in the methylglyoxal-mediated inhibition of p65 DNA-binding. Furthermore, overexpression of p65 inhibited TNF-induced cell death; however, in the presence of exogenously added methylglyoxal, overexpression of p65 caused far greater TNF-induced cell death. These findings suggest that methylglyoxal provides another control mechanism for modulating the expression of NF-κB-responsive genes and that methylglyoxal may be responsible for tipping the balance towards TNF-induced cell death in cells with constitutive NF-κB activation.

Keywords: Abbreviations; MG; methylglyoxal; GLO1; glyoxalase I; TNF; tumor necrosis factor; DMEM; Dulbecco's modified Eagle's medium; PBS; phosphate buffered saline; AGEs; advanced glycation end products; NLS; nuclear localization signalNF-κB; Methylglyoxal; Tumor necrosis factor; Cell death; Necrosis

Lovastatin suppresses erythropoietin receptor surface expression through dual inhibition of glycosylation and geranylgeranylation by Sumaya N. Hamadmad; Raymond J. Hohl (pp. 590-600).

Erythropoietin (Epo) is a cytokine that is required for the survival of erythroid progenitors through interaction with its receptor on the surface of these cells. Recent studies showed that erythropoietin receptor (EpoR) is expressed on many cancer cells. The factors that govern EpoR expression on the cell surface are poorly understood. Using both biotinlyation and radiolabeled Epo binding experiments, we show here that Epo starvation of the Epo-dependent erythroleukemia cell line, ASE2, leads to a time-dependent increase in both forms of EpoR, the maturing 64kDa and the mature 66kDa proteins. Mevalonate depletion inhibits the formation of the highly glycosylated mature form of EpoR without affecting the other form. Treatment of cells with lovastatin, a selective inhibitor of the rate-limiting enzyme in the mevalonate pathway leads to inhibition of cell surface EpoR that is induced by Epo starvation. The effect of lovastatin appears to be the consequence of inhibition of two processes, glycosylation and geranylgeranylation. Adding back geranylgeranyl pyrophosphate to lovastatin-treated cells completely prevents the lovastatin effect on EpoR expression. Dolichol, the sugar carrier in N-linked glycosylation that is derived from the mevalonate pathway, partially reverses lovastatin's effect. The glycosylation inhibitor tunicamycin also partially suppresses EpoR surface expression. Inhibiting protein geranylgeranylation mimics the effect of lovastatin and inhibits EpoR surface expression in a concentration-dependent manner. Finally, lovastatin inhibits Epo's stimulatory effects on cell proliferation. These results indicate that mevalonate derivatives are required for normal EpoR expression on the cell surface through two pathways, glycosylation and geranylgeranylation.

Keywords: Erythropoietin; Erythropoietin receptor; Lovastatin; Geranylgeranylation; Glycosylation; GGPP

Synthesis and pharmacological evaluation of novel β-nitrostyrene derivatives as tyrosine kinase inhibitors with potent antiplatelet activity by Wei-Ya Wang; Pei-Wen Hsieh; Yang-Chang Wu; Chin-Chung Wu (pp. 601-611).

Protein tyrosine kinases have been known to be involved in regulation of platelet aggregation, suggesting a potential target for antiplatelet therapy. Our previous study showed that 3,4-methylenedioxy-β-nitrostyrene (MNS) prevented platelet aggregation caused by various stimulators, and this action was accompanied by inhibition of tyrosine kinases. In the present study, in order to examine the structural determinants required for the actions of MNS and to develop more potent tyrosine kinase inhibitors and antiplatelet agents, a new series of β-nitrostyrene derivatives were synthesized and pharmacologically characterized. The β-nitrostyrene derivatives inhibited thrombin- or collagen-induced human platelet aggregation, ATP secretion, GPIIb/IIIa activation and protein tyrosine phosphorylation. In recombinant enzyme assay, some β-nitrostyrene derivatives also demonstrated potent inhibition of Src and/or Syk kinase activity. Furthermore, there was a good correlation between the inhibitory potency of these compounds on tyrosine kinases and on platelet activation/aggregation. Among them, a benzoyl ester derivative (compound10) possess up to 8-fold greater potency than MNS and over two orders of magnitude greater potency than genistein or tyrphostin A47 in inhibiting platelet responses to thrombin. Our data suggest that β-nitrostyrenes may represent a new class of tyrosine kinase inhibitors with potent antiplatelet activity.

Keywords: Abbreviations; ATP; adenosine 5′-triphosphate; DMSO; dimethyl sulfoxide; FITC; fluorescein isothiocyanate; GF109203X; 3-[1-[3-(dimethylaminopropyl]-1; H; -indol-3-yl]-4-(1; H; -indol-3-yl)-1; H; -pyrrole-2,5-dione monohydrochloride; GP; glycoprotein; MARCKS; myristoylated alanine-rich C kinase substrate; MNS; 3,4-methylenedioxy-β-nitrostyrene; PDBu; phorbol 12,13-dibutyrate; PKC; protein kinase C; RGDS; Arg-Gly-Asp-Ser; SAR; structure–activity relationship; U46619; 9,11-dideoxy-9α,11α-methanoepoxy PGF2; _αβ-nitrostyrene; Protein tyrosine kinase inhibitors; Antiplatelet agents

13-Oxo-ODE is an endogenous ligand for PPARγ in human colonic epithelial cells by Reinhold Altmann; Martin Hausmann; Tanja Spöttl; Michael Gruber; Arthur W. Bull; Katrin Menzel; Daniela Vogl; Hans Herfarth; Jürgen Schölmerich; Werner Falk; Gerhard Rogler (pp. 612-622).

The ligand activated nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ) induces transcriptional repression of pro-inflammatory factors. Activation of PPARγ is followed by amelioration of colitis in animal models of inflammatory bowel disease (IBD). A reduced expression of PPARγ was found in epithelial cells of patients with ulcerative colitis. The eicosanoids 13-HODE and 15-HETE are products of 12/15-lipoxygenase (LOX) and endogenous ligands for PPARγ. Dehydrogenation of 13-HODE by 13-HODE dehydrogenase results in formation of the 13-Oxo-ODE. Highest activity of 13-HODE dehydrogenase is found in colonic epithelial cells (CECs). We therefore investigated whether 13-Oxo-ODE is a new endogenous ligand of PPARγ in CECs.LOX activity and 13-HODE dehydrogenase in CECs were investigated after stimulation with arachidonic or linoleic acid. LOX metabolites were identified by RP-18 reversed-phase HPLC. Binding of¹⁴C-labelled 13-Oxo-ODE was demonstrated using a His-tagged PPARγ.Stimulation of HT-29 and primary CECs homogenates with and without Ca-ionophor was followed by the formation of high amounts of the linoleic acid metabolite 13-Oxo-ODE (155 and 85ng/ml). The decrease of IL-8 secretion from IEC was more pronounced after pre-incubation with 13-Oxo-ODE compared to the PPARγ agonist troglitazone and higher as with the known PPARγ ligands 13-HODE and 15-HETE. Binding assays with¹⁴C-labelled 13-Oxo-ODE clearly demonstrated a direct interaction.High amounts of 13-Oxo-ODE can be induced in CECs by stimulation of linoleic acid metabolism. 13-Oxo-ODE binds to PPARγ and has anti-inflammatory effects. 13-HODE dehydrogenase might be a therapeutic target in IBD.

Keywords: Abbreviations; 5-ASA; 5-aminosalicylic acid; 13-HODE; 13-hydroxyoctadecadienoic acid; 13-Oxo-ODE; 13-oxooctadecadienoic acid; 15-HETE; hydroxyeicosatetraenoic acid; Ab; antibody; CECs; human primary colonic epithelial cells; COX; cyclooxygenase; cPLA; ₂; cytosolic phospholipase A2; 15d-PGJ2; 15-deoxy-Δ12,14-prostaglandin J2; DTT; dithiothreitol; FCS; fetal calf serum; GAPDH; glyceraldehyde-3-phosphate-dehydrogenase; HPLC; high-performance liquid chromatography; IBD; inflammatory bowel disease; IEC; intestinal epithelial cells; IMACs; intestinal macrophages; IL; interleukin; LOX; lipoxygenase; LTB; ₄; leukotriene B; ₄; MHA; 1-hydroxy-8-methoxy-9,10-anthracenedione; MS; mass spectrometry; NF-κB; nuclear factor-kappaB; PCR; polymerase chain reaction; PMSF; phenylmethylsulfonyl fluoride; PPARγ; peroxisome proliferator-activated receptor gamma; RT; reverse transcription; TAB; tetradecyltrimethylammonium bromide; TNBS; trinitrobenzene sulfonic acid; TNF; tumor necrosis factor; TZDs; thiazolidinediones13-Oxo-ODE; Endogenous ligand; 13-HODE dehydrogenase; Ulcerative colitis; PPARγ; Colonic epithelial cells

The ω-atracotoxins: Selective blockers of insect M-LVA and HVA calcium channels by Youmie Chong; Jessica L. Hayes; Brianna Sollod; Suping Wen; David T. Wilson; Peter G. Hains; Wayne C. Hodgson; Kevin W. Broady; Glenn F. King; Graham M. Nicholson (pp. 623-638).

The ω-atracotoxins (ω-ACTX) are a family of arthropod-selective peptide neurotoxins from Australian funnel-web spider venoms (Hexathelidae: Atracinae) that are candidates for development as biopesticides. We isolated a 37-residue insect-selective neurotoxin, ω-ACTX-Ar1a, from the venom of the Sydney funnel-web spider Atrax robustus, with high homology to several previously characterized members of the ω-ACTX-1 family. The peptide induced potent excitatory symptoms, followed by flaccid paralysis leading to death, in acute toxicity tests in house crickets . Using isolated smooth and skeletal nerve-muscle preparations, the toxin was shown to lack overt vertebrate toxicity at concentrations up to 1μM. To further characterize the target of the ω-ACTXs, voltage-clamp analysis using the whole-cell patch-clamp technique was undertaken using cockroach dorsal unpaired median neurons. It is shown here for the first time that ω-ACTX-Ar1a, and its homolog ω-ACTX-Hv1a from Hadronyche versuta, reversibly block both mid–low- (M-LVA) and high-voltage-activated (HVA) insect calcium channel (Ca_v) currents. This block occurred in the absence of alterations in the voltage-dependence of Ca_v channel activation, and was voltage-independent, suggesting that ω-ACTX-1 family toxins are pore blockers rather than gating modifiers. At a concentration of 1μM ω-ACTX-Ar1a failed to significantly affect global K_v channel currents. However, 1μM ω-ACTX-Ar1a caused a modest 18% block of insect Na_v channel currents, similar to the minor block of Na_v channels reported for other insect Ca_v channel blockers such as ω-agatoxin IVA. These findings validate both M-LVA and HVA Ca_v channels as potential targets for insecticides.

Keywords: Abbreviations; ω-ACTX; ω-atracotoxins from Australian funnel-web spiders; BK; _Ca; channel; large conductance calcium-activated potassium channel; Ca; _v; channel; voltage-gated calcium channel; CNS; central nervous system; DUM; dorsal unpaired median; ESI-Q-ToF; electrospray ionization quadrupole time-of-flight; HEPES; N-2-hydroxyethylpiperazine-; N; -2-ethanesulfonic acid; HVA; high-voltage-activated; IC; ₅₀; median inhibitory concentration; ICK; inhibitory cystine-knot; KD; ₅₀; median knockdown dose; K; _v; channel; voltage-gated potassium channel; LD; ₅₀; median lethal dose; M-LVA; mid-low-voltage-activated; MIT; mamba intestinal toxin; Na; _v; channel; voltage-gated sodium channel; rpHPLC; reverse-phase high-performance liquid chromatography; TAG; terminal abdominal ganglion; TFA; trifluoroacetic acid; TEA; tetraethylammonium; (+)-TC; (+)-tubocurarine; TTX; tetrodotoxinω-ACTX-Ar1a; ω-ACTX-Hv1a; ω-Atracotoxin; Voltage-gated calcium channel; Insecticide; Atrax robustus

A novel drug interaction between the quinolone antibiotic ciprofloxacin and a chiral metabolite of pentoxifylline by Jennifer M. Raoul; Marc R. Peterson; Theresa C. Peterson (pp. 639-646).

Pentoxifylline (PTX), a methylxanthine derivative, is metabolized to seven compounds in vivo, with metabolites 1 and 5 possessing biologic activity. Metabolite-1 is a chiral molecule and its S-enantiomer is selectively formed during PTX metabolism in vivo. We have developed a reproducible method of synthesizing a racemic mixture of the chiral metabolite-1 (M-1) of PTX. In this study, we examined the kinetics of racemic M-1 in mice compared to PTX. An interaction between PTX and the quinolone antibiotic ciprofloxacin has been demonstrated. A goal of this study was to determine if a similar interaction occurs between ciprofloxacin and M-1 in vivo. M-1 and PTX had similar absorption and elimination rates. M-1 was rapidly converted to PTX, while very little PTX was converted to M-1 in vivo. The peak concentration of biologically active drug (PTX+M-1) was 36% higher when M-1 was administered compared to PTX. Combination of ciprofloxacin and PTX significantly increased serum concentrations of both PTX and M-1 (2-fold) compared to controls. The combination of M-1 and ciprofloxacin significantly increased serum concentration of M-1 (3-fold) and PTX (2-fold). The ciprofloxacin/M-1 combination produced a significantly higher sera concentration of bioactive drug compared to all other groups suggesting that this combination may enhance the anti-fibrogenic effect.

Keywords: Pentoxifylline; Ciprofloxacin; Metabolite-1; Fibrosis; Metabolism; Drug interaction

Comparative pharmacological analysis of Rho-kinase inhibitors and identification of molecular components of Ca²⁺ sensitization in the rat lower urinary tract by Cleber E. Teixeira; Liming Jin; Fernanda B.M. Priviero; Zhekang Ying; R. Clinton Webb (pp. 647-658).

We aimed to compare the expression and function of molecular components of the RhoA/Rho-kinase signaling pathway in the contractile responses of detrusor, trigonal and urethral smooth muscle, using selective Rho-kinase inhibitors. Contractility studies and molecular approaches were employed to demonstrate the expression patterns and functional activity of the RhoA/Rho-kinase signaling pathway in the lower urinary tract. Frequency–response curves (1–32Hz) and concentration–response curves (CRC) to carbachol (CCh, 0.01–30μM), phenylephrine (PE, 0.01–300μM) and endothelin-1 (ET-1, 0.01–100nM) were significantly attenuated ( p<0.01) following incubation with the Rho-kinase inhibitors H-1152 (0.1–1μM), Y-27632 (1–10μM) or HA-1077 (10μM). Addition of Rho-kinase inhibitors also markedly reduced ( p<0.01) the contractions evoked by either KCl (80mM) or α,β-methylene ATP (α,β-mATP, 10μM). Among the Rho-kinase inhibitors tested, H-1152 was approximately 9–16 times more potent than Y-27632 or HA-1077. In addition, basal tone of detrusor and trigonal strips was reduced following addition of Y-27632 (10μM), H-1152 (1μM) and HA-1077 (10μM). The expression of RhoA, RhoGDI, leukemia-associated RhoGEF (LARG) and p115RhoGEF was similar among the detrusor, trigone and urethra, whereas Rho-kinase α, Rho-kinase β and PDZ-RhoGEF protein levels were significantly lower in the urethra. Components of the RhoA/Rho-kinase signaling are expressed in detrusor, trigonal and urethral smooth muscle and dynamically regulate contraction and tone. Manipulation of RhoGEF expression may provide further understanding of mechanisms involving Ca²⁺ sensitization in the lower urinary tract.

Keywords: Abbreviations; CCh; carbachol; EFS; electrical field stimulation; ET-1; endothelin-1; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GEFs; guanine nucleotide exchange factors; HA-1077; (5-isoquinolinesulfonyl)homopiperazine; H-1152; (; S; )-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine; LARG; leukemia-associated RhoGEF; α,β-mATP; α,β-methylene ATP; MLC; myosin light chain; PE; phenylephrine; RGS; regulator of G protein signaling domain; ROK; Rho-kinase; Y-27632; (; R; )-(+)-; trans; -; N; -(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamideUrethra; Trigone; Detrusor; Rho-kinase; RhoGEFs; RhoA

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Biochemical Pharmacology (v.74, #4)