Kansu Büyükafşar

Expression of Rho-kinase and its functional role in the contractile activity of the mouse vas deferens

The effects of two Rho‐kinase inhibitors, Y‐27632 and fasudil, were investigated on the contractions produced by electrical field stimulation (EFS, 40 V, 1 mS, 2, 4, 8 and 16 Hz, for 20 s), KCl (30 – 60 mM), phenylephrine (Phe) (10−5 – 10−4 M), adenosine‐3′, 5′‐triphosphate (ATP) (10−4 – 10−3 M) and α,β‐methylene ATP (10−5 M). EFS produced frequency‐dependent reproducible contractile activity, which was almost abolished by guanethidine (10−5 M, for 1 h). This contraction consisted of two components (a phasic initial contraction followed by a tonic one), and it was inhibited by Y‐27632 and fasudil (both at 10−5 M). However, these inhibitors had no effect on resting tension of the tissue. Contractions elicited by KCl (30 – 60 mM) were insensitive to guanethidine (10−5 M, for 1 h), but suppressed by Y‐27632 (10−5 M) and fasudil (10−5 M). In addition, the contractions induced by Phe (an α1‐adrenoceptor agonist) and ATP (a purinergic agent) were inhibited significantly by Y‐27632 (10−5 M). Phasic contractions evoked by the selective P2X purinoceptor agonist α,β‐methylene ATP were also suppressed by Y‐27632 (10−5 M). Western blot analysis revealed that the mouse vas deferens expresses Rho‐kinase (ROKα, ROCK‐2 isoform) protein with a molecular weight of approximately 160 kDa. As a positive control, the presence of this protein was also shown in homogenates of smooth muscle from the rat mesenteric artery. In conclusion, Rho‐kinase protein is expressed in the mouse vas deferens, and it mediates neurogenic contractile activity as well as the contractions induced by KCl, Phe, ATP and α,β‐methylene ATP. Owing to the suppressive effects of Rho‐kinase inhibitors on the contractile activity of the vas deferens, the possibility that these compounds might impair ejaculation must be taken into account when considering them as potential agents in the treatment of erectile dysfunction.

Involvement of Rho/Rho-kinase signalling in the contractile activity and acetylcholine release in the mouse gastric fundus.

In this study effects of Rho kinase inhibitors have been examined on the mouse gastric fundal smooth muscle reactivity and neurotransmitter (acetylcholine) release. Two Rho-kinase inhibitors, Y-27632 and fasudil (HA-1077), conspicuously suppressed the contractile responses to carbachol (CCh) and KCl as well as electrical field stimulation (EFS, 40 V, 0.5 ms, and 20 s). pEC(50) value for CCh and EC(50) value for KCl were 6.68+/-0.15 M and 10.4+/-2.8 mM, respectively. EFS induced reproducible contraction (38.3+/-4.75 mN/g tissue) which was almost abolished and potentiated in the presence of atropine (10(-6)M) and eserine (10(-6)M), respectively. The Rho-kinase inhibitors relaxed the fundic strips preconstricted by submaximal concentration of CCh or KCl in a concentration dependent manner. With CCh-elicited contraction, the pEC(50) values of Y-27632 and fasudil were 5.45+/-0.14 and 5.11+/-0.14 M, respectively (p>0.05). However, the pEC(50) values for Y-27632 and fasudil on KCl-induced tone were 6.09+/-0.1 and 5.35+/-0.06 M (p<0.001), respectively. Moreover, [3H]acetylcholine ([3H]ACh) release upon EFS from the gastric fundus was measured and it was found that Y-27632 (10(-4)M) significantly impaired the release. At 3 Hz the radioactivity ratio obtained after and before EFS (S(2)/S(1) ratio) was 0.88+/-0.03 in control but 0.63+/-0.08 in the presence of 10(-4)M Y-27632 (p<0.05). These results suggest that Rho kinase inhibitors can not only relax the gastric fundus but also modulate CCh, cholinergic nerve stimulation, and KCl-induced contraction. Furthermore, Rho/Rho kinase signalling may play a role in the neurotransmitter (ACh) release in the mouse gastric fundus.

Effects of the Rho-kinase inhibitors, Y-27632 and fasudil, on the corpus cavernosum from diabetic mice

Relaxant responses to two Rho-kinase inhibitors, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and fasudil, were compared in the corpus cavernosum obtained from diabetic and non-diabetic mice. Streptozotocin (100 mg kg(-1) day(-1), for 2 days) induced diabetes with a blood glucose level of 318+/-55.4 mg dl(-1); whereas it was 85.4+/-4.1 mg dl(-1) in control mice (P<0.05). Electrical field stimulation (40 V, 0.5 ms, 1, 2, 4, 8, 16 Hz for 15 s) and acetylcholine-induced relaxations were markedly attenuated in the corpus cavernosum from streptozotocin-diabetic mice whereas responses to Y-27632 (10(-9)-3 x 10(-5) M) and fasudil (10(-9)-3 x 10(-5) M) were not altered. EC(50) values for Y-27632 were 2.98+/-0.89 and 4.19+/-2.71 microM in the corpus cavernosum from control and diabetic mice, respectively (P>0.05). The values for fasudil were 7.42+/-4.91 and 3.53+/-1.41 microM in the corpus cavernosum from control and diabetic mice, respectively (P>0.05). These results may suggest that, in diabetes, the relaxant effects of the Rho-kinase inhibitors may not be changed and thus, they may have a beneficial therapeutic effect in diabetic erectile dysfunction.

Expression of Rho‐kinase (ROCK‐1 and ROCK‐2) and its substantial role in the contractile activity of the sheep ureter

Expression of two isoforms of Rho‐kinase (ROCK) and its functional role in the physiological control of smooth muscle contraction in the sheep ureter were investigated. Helical strips of the ureteric smooth muscle were stimulated by electrical field stimulation (EFS, 60 V, 1 mS, 2, 4, 8, 16 and 32 Hz, for 20 S), KCl (80 mM), carbachol (CCh, 10−8–10−4 M) or phenylephrine (Phe, 10−8–10−4 M). EFS produced a reproducible contractile activity, which was abolished by tetrodotoxin (3 × 10−6 M), a Na+ channel blocker. A muscarinic receptor antagonist, atropine (2 × 10−6 M), and an adrenergic neuron blocker, guanethidine (10−5 M), significantly suppressed the contraction induced by EFS. However, this contraction was augmented in the presence of NG‐nitro‐L‐arginine (L‐NA, 10−4 M), a nitric oxide synthase inhibitor. Two Rho‐kinase inhibitors, Y‐27632 (5 × 10−5 M) and fasudil (5 × 10−5 M), markedly attenuated the EFS‐elicited contraction. CCh and Phe produced concentration‐dependent contraction in the sheep ureter. pD2 values for Phe and CCh were 5.04±0.11 and 5.00±0.22, respectively. Y‐27632 (5 × 10−5 M) and fasudil (5 × 10−5 M) also significantly inhibited CCh‐ and Phe‐induced contractions. Moreover, these ROCK inhibitors produced relaxations in the KCl‐elicited contraction in a concentration‐dependent manner. pD2 values for Y‐27632 and fasudil were, respectively, 5.17±0.07 and 4.58±0.08 (P<0.001). Furthermore, the influences of these agents were also tested on spontaneous phasic contractions of the tissue. Among Y‐27632, fasudil, TTX, L‐NA, guanethidine and atropine, only the ROCK inhibitors (10−6–10−5 M) were able to suppress the spontaneous contractile activity. Western blot analysis has revealed that both isoforms of Rho‐kinase (ROCK‐1 and ROCK‐2) are expressed in the sheep ureter. Densitometric analysis has indicated that these enzymes are less expressed in the sheep ureter than are in the sheep aorta in a significant manner. These results show that a contractile enzyme, Rho‐kinase, is expressed, and it mediates agonist‐ and EFS‐induced contractions as well as spontaneous contractile activity of the isolated sheep ureter. Since Y‐27632 and fasudil depressed the contractions, it seems plausible to postulate that Rho‐kinase inhibitors may be beneficial in the treatment of renal colic.

Antiepileptic effects of two Rho‐kinase inhibitors, Y‐27632 and fasudil, in mice

Rho/Rho‐kinase signalling is involved in many cellular events, including some in the CNS. However, the role of this pathway in epilepsy has not yet been assessed. Therefore, we determined the effects of two Rho‐kinase inhibitors, Y‐27632 and fasudil, on seizures induced by pentylenetetrazole (PTZ) or maximal electroconvulsive shock (MES).

Nebivolol, but Not Metoprolol, Improves Endothelial Function of the Corpus Cavernosum in Apolipoprotein E-Knockout Mice

To determine the effects and underlying mechanisms of treatment with the β-receptor blockers nebivolol and metoprolol on penile endothelial function in apolipoprotein E (ApoE)-/- mice, wild-type (WT) and ApoE-/- mice were fed with a cholesterol-rich diet for 7 weeks. ApoE-/- mice were treated with nebivolol (10 mg/kg/day) or metoprolol (90 mg/kg/day). Endothelial function of aortic and corpora cavernosal tissue was assessed by pharmacological stimulation with carbachol (endothelium dependent) or glycerol trinitrate (endothelium independent) in organ bath experiments. Atherosclerotic lesion formation was evaluated with oil-red staining, and modulation of reactive oxygen species (ROS) production was determined with lipid peroxidation. Heart rate, but not blood pressure, was decreased in nebivolol- and metoprolol-treated ApoE-/- mice (p < 0.01) compared with controls and WT mice without significant intergroup differences. Atherosclerotic lesion formation in the aortic root was increased in ApoE-/- mice (p < 0.01) with a more significant improvement in nebivolol- (p < 0.01) compared with metoprolol-treated mice (p < 0.05). Endothelium-dependent relaxation of the corpora cavernosa was significantly impaired in ApoE-/- mice (p < 0.05), which improved in nebivolol- versus metoprolol-treated mice. Efficacy of endothelium-dependent relaxation was comparable in aortic and penile tissue. Quantification of ROS production via lipid peroxidation revealed a significant reduction of superoxide anion production in nebivolol-treated (p < 0.05) but not metoprolol-treated mice compared with ApoE-/- controls. Nebivolol improves penile endothelial function as a surrogate of erectile function in ApoE-/- mice. These effects may be related to a reduction of ROS production, which is independent of heart rate reduction, because metoprolol did not increase endothelial function.

Oxidation of nitroxyl anion to nitric oxide by copper ions

This study made use of a nitric oxide‐sensitive electrode to examine possible means of generating nitric oxide from nitroxyl anion (NO−) released upon the decomposition of Angelis salt. Our results show that copper ions (from CuSO4) catalyze the rapid and efficient oxidation of nitroxyl to nitric oxide. Indeed, the concentrations of copper required to do so (0.1–100 μM) are roughly 100‐times lower than those required to generate equivalent amounts of nitric oxide from S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP). Experiments with ascorbate (1 mM), which reduces Cu2+ ions to Cu+, and with the Cu2+ chelators, EDTA and cuprizone, and the Cu+ chelator, neocuproine, each at 1 mM, suggest that the oxidation is catalyzed by copper ions in both valency states. Some compounds containing other transition metals, i.e. methaemoglobin, ferricytochrome c and Mn(III)TMPyP, were much less efficient than CuSO4 in catalyzing the formation of nitric oxide from nitroxyl, while FeSO4, FeCl3, MnCl2, and ZnSO4 were inactive. Of the copper containing enzymes examined, Cu‐Zn superoxide dismutase and ceruloplasmin were weak generators of nitric oxide from nitroxyl, even at concentrations (2500 and 30 u ml−1, respectively) vastly greater than are present endogenously. Two others, ascorbate oxidase (10 u ml−1) and tyrosinase (250 u ml−1) were inactive. Our findings suggest that a copper‐containing enzyme may be responsible for the rapid oxidation of nitroxyl to nitric oxide by cells, but the identity of such an enzyme remains elusive.

THE ROLE OF POLY(ADP-RIBOSE) SYNTHETASE INHIBITION IN PREVENTING ENDOTOXEMIA-INDUCED INTESTINAL EPITHELIAL APOPTOSIS

In this lipopolysaccharide (LPS)-induced endotoxemia model, the effects of 3-aminobenzamide (3-AB), a poly(ADP-ribose) synthetase (PARS) inhibitor, on ileal apoptosis were evaluated by light microscopy and M30 cell death staining. Moreover, the relationship between Bcl-2, iNOS expression, and serum nitrate (NO(3)(-)) levels were investigated. Thirty-two male Wistar rats, weighing 180-220g were randomly divided into four groups. The group I (control; n=8) received saline and group II (sepsis; n=8) received 10 mg kg(-1) LPS intraperitoneally. 3-AB was given to the group IV (S+3-AB; n=8) 20 min before giving LPS and to the group III (C+3-AB; n=8) 20 min before giving saline. Six hours later, blood and ileum samples were taken. Endotoxemic group exhibited significant apoptosis in intestinal epithelial cells and the immunohistochemical examination with M30 was demonstrated that the 3-AB reduced the LPS-induced intestinal apoptosis. Serum NO(3)(-) level was increased in endotoxemic group, whereas the elevation of NO(3)(-) level was prevented in LPS+3-AB group (P<0.05). The increased iNOS expression observed in the LPS group was also prevented by 3-AB. Compared with the endotoxemic group, ileal epithelial columnar cells from LPS+3-AB group had a dense Bcl-2 staining which was almost identical with control. In conclusion, 3-AB decreases LPS-induced apoptosis in ileum by preventing LPS-induced depletion of Bcl-2 and blocking iNOS gene. Modification of Bcl-2 expression by PARS inhibitors should further be investigated as a new therapeutic alternatives in septic states.

Protective Effect of L-Arginine Intake on the Impaired Renal Vascular Responses in the Gentamicin-Treated Rats

The purpose of this study was to investigate the effect of gentamicin (100 mg/kg/day, i.p.) treatment on endothelium-dependent and -independent vasodilation in isolated perfused rat kidney, and the effect of amino acid L-arginine (in the drinking water, 2.25 g/l) on renal dysfunction induced by gentamicin. When gentamicin-treated groups were compared with the control group, it was observed that BUN and creatinine levels increased significantly. Also, the relaxant responses induced by acetylcholine, sodium nitroprusside and pinacidil decreased. Histopathological examination indicated acute tubular necrosis in this group. In animals treated with gentamicin together with L-arginine, there was a significant amelioration in the BUN and creatinine levels. The vasodilator responses were similar to those of the control group. Histopathological examination indicated only hydropic degeneration in tubular epithelium of kidney. Co-administration of L-NG-nitroarginine methyl ester (L-NAME) (112.5 mg/l), an inhibitor of nitric oxide synthase, and L-arginine to rats treated with gentamicin did not change the protective effect of L-arginine. In rats receiving L-NAME alone, the level of BUN and creatinine and vasodilation to acetylcholine were not significantly different when compared to those of the control group, while relaxant responses to sodium nitroprusside and pinacidil were increased. These results suggest that gentamicin leads to an impairment in vascular smooth muscle relaxation in addition to acute tubular necrosis in the rat kidney. Supplementation of L-arginine has an important protective effect on gentamicin-induced nephropathy.

Vasoconstriction induced by G1, a G-protein-coupled oestrogen receptor1 (GPER-1) agonist, in the isolated perfused rat kidney.

Vascular effects of the G protein-coupled oestrogen receptor1 (GPER-1) agonist, G1 (10(-7)-5×10(-6) M), the main oestrogenic hormone, 17β-estradiol (10(-9)-10(-4) M), the NR3A1 agonist, PPT (10(-8)-10(-5) M), the NR3A2 agonist DPN (10(-8)-10(-5) M), and the classical oestrogen receptor blocker but also a GPER agonist, ICI-182780 (10(-8)-3×10(-6) M), were investigated on the perfusion pressure in the isolated rat kidney. To seek cellular mechanisms involved in GPER-1-induced signalling we tested several compounds including the inhibitors of Rho-kinase (ROCK) (Y-27632), tyrosine kinase (genistein), p38MAPK (SB203580), p44/42MAPK (PD98059), protein kinase C (PKC) (GF109203X), Jun-kinase (JNK) (SP600125), phosphatidylinositol-3-kinase (PI3K) (LY294002), Ca(2+) channels (nifedipine), GPER-1 (G15) and epidermal growth factor (EGF) receptor kinase (AG-1478). Moreover, the effect of saponin (50mg/ml) that was used for endothelium removal was explored on G1-elicited vascular action. G1, 17β-estradiol and ICI-182780 but not PPT and DPN induced vasoconstrictions in basal renal perfusion pressure. In contrast, G1 promoted vasodilatation when the perfusion pressure was elevated in advance by phenylephrine. G1-elicited vasoconstriction was not modified by endothelial removal; however, it was markedly inhibited by GPER-1 antagonist, G15. The vasoconstrictor response to G1 was also significantly attenuated by Y-27632, PD98059, SB203580, GF109203X, genistein, AG-1478, and nifedipine, but not LY294002 and SP600125. Western blotting indicated the expression of GPER-1 in renal artery, medulla and cortex of rat kidney. In conclusion, GPER-1 could substantially modulate vascular responses through a variety of signalling pathways including ROCK, PKC, p38 MAPK, p42/44 MAPK, tyrosine kinase, EGF receptor kinase and VOCC but not JNK or PI3K in isolated perfused rat kidney.