Search

Background Rho/ROCK signaling has been demonstrated to be involved in the vascular reactivity of many arterial networks. However, RhoA expression and the contribution of Rho/ROCK pathway to the control of perfusion pressure have not been investigated in the rat hind limb vascular bed as a skeletal muscle vascular network. Aims To investigate the contribution of the Rho/ROCK pathway in the control of perfusion pressure in the isolated-perfused rat hind limb vascular bed. Study design Animal experimentation. Methods Two Rho inhibitors (atorvastatin and C3 exoenzyme) and ROCK inhibitors (Y-27632 and fasudil) were tested on the phenylephrine-elevated perfusion pressure in the isolated-perfused rat hind limb vascular bed. Furthermore, we sought the expression of RhoA protein in the femoral, popliteal and saphenous arteries as well as quadriceps and gastrocnemius muscles by Western blotting. Results The ROCK inhibitors Y-27632 and fasudil (both 10-8 to 10-5 M) induced substantial vasodilatations. The maximum vasodilatations induced by Y-27632 and fasudil (both at 10-5 M) were 84.0 ± 6.9% and 76.9 ± 6.9%, respectively (P = .091). Y-27632 was not more potent than fasudil, as the EC50 values for Y-27632 and fasudil were 0.7 ± 2.1 μM and 2.5 ± 2.4 μM, respectively (P = .177). Atorvastatin (10-7 to 10-4 M) and C3 exoenzyme (3 × 10-8 M) also produced vasodilatation (maximum vasodilatation; 20.3 ± 1.7% and 13.7 ± 3.6%, respectively). The EC50 value for atorvastatin was 94.9 ± 1.2 μM. The western blot analysis showed that the femoral, saphenous, and popliteal arteries, as well as the gastrocnemius and quadriceps muscles, express RhoA protein. Conclusion The Rho/ROCK pathway contributes significantly to the control of perfusion pressure in the rat hind limb vascular bed.

Background/aim The effect of testosterone replacement therapy was investigated on bladder functions, histology, apoptosis as well as Rho-kinase expression in the rat bladder outlet obstruction (BOO) and hypogonadism models. Materials and methods 30 mature male rats divided into 4 groups: sham group (n = 8), BOO group (n = 8), BOO + orchiectomy group (n = 7), BOO + orchiectomy + testosterone (T) treatment group (n = 7). Cystometric findings, apoptosis index, Rho-kinase (ROCK-2) expression, and smooth muscle/collagen ratio were compared. Results BOO did not change ROCK-2 expression level, compared to sham group (P > 0.05). However, when compared to BOO group (P < 0.01), BOO + orchiectomy led ROCK-2 increase. The testosterone treatment failed to reverse the up-regulation of ROCK-2 induced by orchiectomy although it tended to lower ROCK-2 level. Compared to sham group (P = 0.002), changes in maximal bladder capacity and leak point pressure were higher (P = 0.026, P = 0.001), and bladder compliance was lower in BOO group. Also, the apoptosis index was different between the two groups (P = 0.380). Smooth muscle/collagen ratio was higher in BOO + orchiectomy + T group than in BOO + orchiectomy group (P = 0.010). Conclusions The research draws attention to alternating treatment approaches in case of the presence of hypogonadism and BOO.

Objectives: The most diagnosed tumor among infiltrating tumors of the female genital tract is endometrial carcinoma. The Wnt/β-catenin signaling pathway has an important role in organogenesis, self-renewal of tissues, and adult stem cell maintenance. However aberrant activation of it causes many types of tumors and also related to the prognosis of patients. Therefore, we aimed to investigate whether Wnt/β-catenin pathway inhibitors have any effect on the proliferation and migration of tumor cells. Methods: As cancer cell line, HEC-1A endometrial adenocarcinoma was used. The Wnt/β-catenin pathway inhibitors effects on proliferation and migration were demonstrated by real-time cell analysis device and wound healing model respectively. Results: Wnt/β-catenin pathway inhibitors FH535 (25 μM at 36th hour, p < 0.05; 50μM at 48th hour p < 0.001) and niclosamide inhibited cell proliferation (10, 25 and 50μM at 60th hours; p < 0.01, p < 0.05 and p < 0.001, respectively) whereas ICRT14 and IWP-2 did not. However only niclosamide which is also an antihelmintic drug inhibited migration of the cells in all concentrations tested (10, 25 and 50μM, p < 0.05). Conclusions: The present study shows that the Wnt/β-catenin pathway has an substantial role in both proliferation and migration of endometrial adenocarcinoma. We suggest that the antihelmintic drug niclosamide could be further investigated for its potential therapeutic effect in endometrial adenocarcinoma.

Amaç: Bu çalışmada Mersin Üniversitesi Hastanesi’nde çalışan araştırma görevlilerinin akılcı ilaç kullanımı (AİK) konusunda bilgi ve yaklaşımlarını belirlemek amaçlanmıştır. Yöntem: Kesitsel ve tanımlayıcı tipte olan bu çalışmada Mersin Üniversitesi Hastanesi’nde çalışmakta olan araştırma görevlilerine demografik verilere ait 6 soru ve akılcı ilaçla ilgili bilgi düzeylerini ve yaklaşımlarını ölçmek üzere hazırlanan 46 soru olmak üzere toplam 52 sorudan oluşan bir anket uygulanmıştır. Bulgular: Bu çalışmaya Mersin Üniversitesi Hastanesi’nden 180 kişi dahili, 69 kişi cerrahi bölümlerden olmak üzere 249 araştırma görevlisi katıldı. 169’u (%67.9) daha önce AİK ile ilgili eğitim almış 80’i ise (%32.1) herhangi bir eğitim almamış olan katılımcıların %78.1’i tıp eğitimi sırasında, %22.5’i mezuniyet sonrası ve %43.8’i de asistanlık süreçlerinde teorik eğitimler aldıklarını ifade ettiler. Hastanın yaşını, kullanmakta olduğu diğer ilaçları ve kronik hastalıklarını soranlar ise sırası ile katılımcıların%96.8, %94.8ve %98.0’i dir. Hastanın gebeliğini sorgulayanlar katılımcıların %87.1 ve emzirip emzirmediğini soranlar ise %85.5'i idi. Katılımcıların sadece %67.9’u hastasına yazdığı ilacın adını söylerken, ilaca ait yan etkilerden hastaya bahsetmeyenlerin sadece %30’u AİK ile ilgili eğitim almayanlardı. Öte yandan katılımcıların %25.5’i ilaç seçerken maliyet kriterini hiç dikkate almazken etkinlik, güvenlik ve uygunluğu daha fazla önemsemekteydiler. Maliyeti dikkate almayanlardan ise akılcı ilaç konusunda eğitim almayanların oranı ise %39.7 idi. Sonuç: Araştırma görevlilerinin daha önce eğitimler almış olsalar da AİK konusunda gerek bilgi düzeyleri gerek se tutum ve yaklaşımları açısından geliştirilmesi gereken noktalar olduğu tespit edilmiştir. Bu nedenle AİK ile ilgili süre ve içerik açısından zenginleştirilmiş, özellikle de uygulamalı eğitimlerin yapılması akılcı ilaç reçeteleme davranışı ile ilgili tespit edilen eksikliklerin giderilmesi yönünde önemli katkılar sağlayacaktır., Aim: In this study, it was aimed to determine the knowledge and approaches of research assistants working in Mersin University Hospital on rational use of drug (RDU). Method: In this cross-sectional and descriptive study, a questionnaire consisting of 52 questions in total, including 6 questions on demographic data and 46 questions prepared to measure their knowledge and approaches about rational medicine, was applied to the research assistants working at Mersin University Hospital. Results: 249 research assistants from Mersin University Hospital, including 180 internal and 69 from the surgical departments, participated in this study. Participants, 169 (67.9%) of whom had previously received training on RDU and 80 (32.1%) had not received any training stated that they received theoretical training on rational drug use during medical education (78.1%), after graduation (22.5%) and during assistantship (43.8%). Those asking about the age of the patient, the other medications he is using, and his chronic diseases are 96.8%, 94.8% and 98.0% of the participants, respectively. Those questioning the pregnancy of the patient were 87.1% of the participants and 85.5% of them asked whether she was breastfeeding or not. While only 67.9% of the participants said the name of the drug they prescribed to their patients, only 30% of those who did not mention the side effects of the drug to the patient were those who did not receive training on RDU. On the other hand, while 25.5% of the participants did not consider the cost criteria when choosing drugs, they gave more importance to efficiency, safety and suitability. 39.7% of those who did not consider the cost did not receive any training about RDU. Although the research assistants have received training before, it has been determined that there are points that need to be improved in terms of both their level of knowledge and their attitudes and approaches on RDU. Conclusion: Although the research assistants have received training before, it has been determined that there are points that need to be improved in terms of both their level of knowledge and their attitudes and approaches on RDU. For this reason, enriched courses in terms of duration and content, and especially practical training on RDU will provide significant contributions to overcome the deficiencies identified in rational drug prescribing behavior.

Endometrial cancer is the most common gynecological cancer in developed countries. Potassium channels, which have many types, are suggested to play a major role in cancer progression. However, their role in endometrial cancer has not been fully investigated. We aimed to demonstrate whether the ATP-sensitive potassium channel blocker glibenclamide, voltage-sensitive potassium channel blocker 4-aminopyridine, non-selective (voltage-sensitive and calcium-activated) potassium channels blocker tetraethylammonium and potassium chloride (KCl) have any effect on the proliferation and migration of HEC1-A cells.Proliferation and migration were evaluated by real-time cell analysis (xCELLigence system) and wound healing assays, respectively. Proliferation was reduced by glibenclamide (0.1 and 0.2 mM, P 0.05 and P 0.01, respectively), 4-aminopyridine (10 and 20 mM, P 0.001) and tetraethylammonium (10 and 20 mM, P 0.01 and P 0.001, respectively). However, KCl did not change the proliferation. Migration was reduced by glibenclamide (0.01, 0.1 and 0.2 mM, P 0.001, P 0.001 and P 0.01, respectively) and 4-aminopyridine (10 and 20 mM, P 0.05 and P 0.01, respectively). Tetraethylammonium did not change migration. However, KCl reduced it (10, 25 and 50 mM, P 0.05, P 0.01 and P 0.01, respectively). Both proliferation and migration were reduced by glibenclamide and 4-aminopyridine. However, tetraethylammonium only reduced proliferation and KCl only reduced migration.Potassium channels have an important role in HEC1-A cell proliferation and migration and potassium channel blockers needs to be further investigated for their therapeutic effect in endometrial cancer.

It has been clearly indicated that osteoarthritis (OA) is an inflammatory and degenerative disease that could be promoted by Rho-kinase (ROCK); however, little is known about the role of ROCK/inhibitor κB alpha (IκB-α)/nuclear factor-κB (NF-κB) p65 pathway activation in interleukin-1β (IL-1β) induced inflammatory response and oxidative stress in primary human chondrocytes. To test this hypothesis, we focused on determining ROCK-II, IκB-α, p-IκB-α, NF-κB p65, p-NF-κB p65, IL-6, tumor necrosis factor alpha (TNF-α), cyclooxygenase-2 (COX-2), p22phox, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subtype 4 (NOX4) protein expression, ROCK-II activity, NADPH oxidase levels, and total antioxidant capacity (TAC) in the presence and absence of ROCK-inhibitor fasudil. IL-1β (2 ng·mL–1, 24 h) increased the expression of ROCK-II, p-IκB-α, NF-κB p65, p-NF-κB p65, IL-6, TNF-α, COX-2, and p22phox proteins, and decreased the expression of IκB-α, and the NOX4 protein level did not alter. ROCK activity and NADPH oxidase levels were increased, whereas the TAC was decreased by IL-1β. Fasudil (10−5–10−7 M) reversed all these changes induced by IL-1β. These results demonstrate that ROCK/IκB-α/NF-κB p65 pathway activation contributes to the IL-1β-induced inflammatory response and oxidative stress, and thus, ROCK inhibition might be a beneficial treatment option for OA patients mainly based on its anti-inflammatory and antioxidant effects.

Aim:Although detection and monitoring of adverse drugreactions is mandatory for drug safety, the reporting of adverse drug reactionsare insufficient in our country. On the other hand, raising awareness of theimportance of pharmacovigilance among nurses is important for reporting adversedrug reactions. The main purpose of this study is to evaluate the knowledge,attitudes and practices about pharmacovigilance of nurses working in auniversity hospital.Methods:In this cross-sectional and descriptive study, a25-question questionnaire was applied to evaluate their knowledge, attitudesand practices on pharmacovigilance to nurses working in Mersin UniversityHospital.Results:83.9% of the nurses defined the pharmacovigilancecorrectly and 40.7% knew that there was a pharmacovigilance contact point inthe hospital. 81.7% of nurses are aware that reporting adverse drug reactionsis a professional obligation. While 48.8% of the nurses reported that they hadexperienced an adverse drug reaction but only 0.2% of them had reported anadverse drug reaction. 75.1% of the nurses reported that it was difficult todecide on an adverse drug reactionand 36.6% of the nurses showed that there was not enough time to report anadverse drug reaction.Conclusions:In this study, it was observed that nursesparticipating in the study had deficiencies in knowledge, attitude andpractices related to pharmacovigilance. The rate of adverse drug reactionreporting among nurses is very low. Pharmacovigilance training should beorganized for nurses in hospitals and nurses should be involved in thisprocess. In this way, it could be ensured that nurses make the necessarycontribution to the reporting of adverse drug reactions., Amaç: Advers ilaç reaksiyonlarının tespit edilmesi ve izlenmesi ilaçgüvenliği açısından zorunlu olmasına rağmen, ülkemizde advers ilaç reaksiyonubildirimi yetersizdir. Öte yandan hemşirelerde farmakovijilansın önemi hakkındafarkındalık yaratmak advers ilaç reaksiyonu bildirimi için önem arz etmektedir.Bu çalışmanın temel amacı bir üniversite hastanesinde çalışan hemşirelerinfarmakovijilans konusundaki bilgi, tutum ve uygulamalarını değerlendirmektir.Yöntem: Kesitsel ve tanımlayıcı tipte yapılan bu çalışmada, MersinÜniversitesi Hastanesinde çalışan hemşirelerin farmakovijilans konusunda bilgi,tutum ve uygulamalarını değerlendirmek için 25 soruluk anket uygulanmıştır. Bulgular: Hemşirelerin %83,9’u farmakovijilansı doğru tanımlamış ve %40,7’sihastanede bir farmakovijilans irtibat noktasının olduğunu bilmiştir.Hemşirelerin %81,7’si advers ilaç reaksiyonu bildiriminin profesyonel biryükümlülük olduğunun farkındadır. Hemşirelerin %48,8’i yaşanan bir advers ilaçreaksiyonu gördüğünü bildirmekle birlikte sadece %0,2’si advers ilaç reaksiyonubildirimi yaptığını ifade etmiştir. Yaşanan bir advers ilaç reaksiyonunutanımlamada hemşirelerin %75,1’i karar vermenin zor olduğunu bildirmiş veadvers ilaç reaksiyonunu bildirmek için hemşirelerin %36,6’sı yeterlizamanlarının olmadığını sebep olarak göstermişlerdir.Sonuç: Bu çalışmada araştırmaya katılan hemşirelerin farmakovijilans ileilgili bilgi, tutum ve uygulamalarında eksiklikleri olduğu görülmüştür.Hemşireler arasında advers ilaç reaksiyonu bildirimi oranı çok düşüktür.Hastanelerde hemşireler için farmakovijilans ile ilgili eğitimler düzenlenmelive hemşirelerin bu süreçte yer almaları sağlanmalıdır. Bu şekilde advers ilaçreaksiyonlarının bidiriminde hemşirelerin gerekli katkıyı sunması sağlanabilir.

Effect of female sex hormones on the production/release of adipocyte-derived cytokines has been debatable. Furthermore, whether the cellular signaling triggered by these hormones involve Rho-kinase has not been investigated yet. Therefore, in this study, effects of 17β-estradiol and progesterone as well as the Rho-kinase inhibitor, Y-27632 on the level of adipokines such as resistin, adiponectin, leptin, TNF-α and IL-6 were investigated in 3T3-L1-derived adipocytes. Differentiation was induced in the post-confluent preadipocytes by the standard differentiation medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum together with the mixture of isobutylmethylxanthine, dexamethasone and insulin) in the presence of 17β-estradiol (10(-8)-10(-7)M), progesterone (10(-6)-10(-5)M), the Rho-kinase inhibitor, Y-27632 (10(-5)M) and their combination for 8days. Measurements of the adipokines were performed in the culturing medium by ELISA kits using specific monoclonal antibodies. 17β-estradiol elevated resistin but decreased adiponectin and IL-6 levels; however, it did not alter the concentration of leptin and TNF-α. Y-27632 pretreatment inhibited the rise of resistin and the fall of adiponectin by 17β-estradiol without any effects by its own. Progesterone did not change resistin, leptin and TNF-α level; however, it elevated adiponectin and decreased IL-6 production. Neither 17β-estradiol nor Y-27632 was able to antagonize the increase of adiponectin and the reduction of IL-6 levels by progesterone. While Y-27632 alone lowered IL-6 level, it increased leptin and TNF-α concentration without altering resistin and adiponectin. In conclusion, 17β-estradiol could modify adipokine production in 3T3-L1 adipocytes with the actions some of which involve Rho-kinase mediation.

Aim: Chronic myeloid leukemia (CML) is a BCR-ABL positive myeloproliferative disorder characterized by clonal proliferation of the hematopoietic stem cells. Imatinib mesylate represents the most important agent associated with improved survival in CML that has been introduced for clinical use in 2000s. The original imatinib preparation Glivec ® was subsequently followed by the introduction of biosimilar products containing a variety of excipients. Gastrointestinal side effects represent the major limitation for therapeutic use of imatinib. This study was planned to examine whether excipients in the original and biosimilar imatinib products had any role in the emergence of GIS side effects. Materials and methods: Excipients in imatinib preparations used for the treatment of chronic phase (CP) CML patients followed up and treated at the hematology department of Mersin University between 2000 and 2019 were analyzed with respect to their potential GIS side effects. Results: Totally 42 CML patients included in the study. The median age was 53.2, and female:male ratio was 20:22. They had similar demographic characteristics as compared to previously reported CML populations. No patients had GIS side effect requiring drug discontinuation or switch to another agent. While bovine gelatin and polyvinyl alcohol had no significant effects on GIS side effects, those that contained titanium were found to be associated with a significant increase in the risk of GIS side effects (52%, contained titanium vs 0/2, contained no titanium). These side effects are less frequently in products that have lowest number of excipient types. Conclusion: GIS side effects can be triggered by various excipients in imatinib preparations.

objective. Effect of estrogenic compounds and 17β-estradiol (E2), which induces endothelial cell motility, was investigated on ROCK-2 expression in rat coronary vascular endothelial cells (CVEC). Methods. The CVEC were isolated from the heart of Wistar rats by collagenase (0.04%) and incubated with E2 (1-100 nM), estrogen receptor α (ERα) agonist: propyl pyrazole triol (PPT, 10 nM); ERβ agonists: (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN, 10 nM) and E2-conjugate with bovine serum albumin (E2-BSA, 1 nM); and GPER1 agonist: G1 (100 nM). Furthermore, the effect of combination of E2 with estrogen receptors (ERs) antagonist and GPER1 agonist, ICI-182780 (10 µM), physiological estrogen antagonists: progesterone (P4, 10-100 nM) and testosterone (T, 10-100 nM); transcription inhibitor: actinomycin-D (1 µg/ml); GPER1 antagonist: G-15 (100 nM), superoxide dismutase, (SOD, 500 U/ml); G i/o protein inhibitor: pertussis toxin (PTX, 100 µg/ml); and epidermal growth factor receptor (EGFR) blocker: AG-1478 (10 µM) was tested. After 24h incubation, ROCK-2 and GPER1 protein expressions were detected in the CVEC by Western-blotting. results. E2, ICI-182780, and G1 but not E2-BSA significantly up-regulated ROCK-2 expression, which was suppressed by actinomycin-D, PTX, AG-1478, and G-15. However, PPT and DPN had no effects on the ROCK-2 expression. ICI-182780, P4, T or SOD did not antagonize the E2 action. GPER1 expression was demonstrated in the CVEC. Conclusions. Estrogens could up-regulate ROCK-2 in the rat CVEC through GPER1 and EGFR transactivation.

This study aimed to determine the effect of pluripotent astrocytic stem cells (PASCs) and fibroblast growth factor-2 (FGF-2) on cognitive function in neonatal rats with hypoxic-ischemic brain injury (HIBI).The study was performed on 7-d-old rats that were randomly divided into four groups. All rats, except those in the sham group, were kept in a hypoxic chamber containing 8% oxygen for 2 h after the ligation of the right carotid artery. Next, 5 d after HIBI was induced, PASCs were administered to the motor cortex, and FGF-2 was administered intraperitoneally to group AF; PASCs were administered to the motor cortex, and salt solution buffered with phosphate was administered intraperitoneally to group A; and fresh cell culture solution (medium) was administered to group M. Immunofluorescence was used to localize the administered PASCs in the brains of rats from groups A and AF. The Morris water maze tank (MWM) test was performed to assess the rats' cognitive functions at week 12. The rats that were administered PASCs were observed for the development of neoplasms and autopsies were performed after 30 months.PASCs migrated to damaged brain regions surrounding the hippocampus in groups A and AF. The mean platform finding time (PFT) significantly decreased over time in each group on day 1-4 of MWM testing (p 0.001). On day 2-4, the mean PFT was shortest in group S followed by group AF. In group A, the PFT was significantly longer than in group S on day 3-4 (p = 0.01 and 0.007, respectively). On day 5 of the MWM test, the time spent in the eastern quadrant (which previously contained the platform) was longest in group S followed by groups AF, A, and M; however, the differences between groups were not significant (p = 0.51). After 30 months, none of the rats in groups A or AF had benign or malignant neoplasms.Following the administration of PASCs in rats with experimentally induced HIBI, PASCs migrated to the injured brain regions; however, treatment with PASCs did not have a positive effect on cognitive function. The administration of FGF-2 together with PASCs resulted in positive cognitive results, although not at the level of significance.

Rho kinase has contractile activity, which induces Ca2+ sensitization in various cells. Several receptors are linked to the Rho/Rho-kinase pathway. Therefore, in this study we aimed to demonstrate the central importance of this novel pathway for diverse excitatory stimuli in the smooth muscle of the sheep gallbladder. Accordingly, the effects of a Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10(-8)-3 x 10(-5) M), were investigated on cholecystokinin-8 (CCK-8, 10(-8) M), endothelin-1 (10(-8) M), carbachol (10(-6)-10(-5) M), 5-hydroxytryptamine (5-HT, 10(-6)-10(-5) M), histamine (10(-6)-10(-5) M), phenylephrine (10(-5)-10(-4) M), neurokinin A (10(-7)-10(-6) M), electrical field stimulation (40 V, 0.5 ms, 2, 4, 8, 16, 32 Hz, 15 s, 3 min intervals) and potassium chloride (KCl, 25-50 mM)-induced contractions as well as spontaneous contractile activity. Electrical field stimulation evoked tetrodotoxin (3 x 10(-6) M)-sensitive reproducible contractions, which were inhibited by atropine (2 x 10(-6) M) and potentiated by eserine (5 x 10(-7) M). EFS-induced contraction was significantly inhibited by Y-27632 (10(-5) M). In addition, spontaneous contractile activity was suppressed in the presence of the compound (10(-6)-10(-5) M). This Rho kinase inhibitor also dramatically decreased the contractions elicited by 5-HT, neurokinin A and carbachol. KCl-induced contraction, which was not atropine-sensitive, was also conspicuously attenuated by Y-27632. Moreover, Y-27632 (10(-8)-3 x 10(-5) M) relaxed gallbladder strips that were contracted by histamine, endothelin-1, CCK-8 and phenylephrine in a concentration-dependent manner. pEC50 values for Y-27632 were 6.25+/-0.10, 5.79+/-0.12, 5.83+/-0.09 and 5.70+/-0.13 for the contraction elicited by histamine, CCK-8, endothelin-1 and phenylephrine, respectively. Furthermore, we also demonstrated Rho kinase protein expression (ROCK-1 and ROCK-2) by Western blot analysis. In conclusion, ROCK is expressed in the smooth muscle of the ovine gallbladder, and it has a central role in the contractile activity induced by diverse excitatory stimuli.

The possible contribution of Rho/Rho-kinase signalling in oleic acid (100 mg kg −1 , i.v., for 4 h)-induced lung injury was investigated in rats. Furthermore, the possible protective effect of the administration of a Rho-kinase inhibitor, (+)-( R )- trans -4-(1-aminoethyl)- N -(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 0.5–5 mg kg −1 , i.v., 15 min before the administration of oleic acid), was also examined. Western blot analysis as well as histopathological examination revealed that Rho-kinase (ROCK-1 and ROCK-2) was upregulated in lungs obtained from oleic acid-administrated rats. In addition, the markers of oxidative and nitrosative stress, i.e., malondialdehyde, myeloperoxidase, 3-nitro- l -tyrosine and nitrite/nitrate, in serum and lung tissue were also increased in the injury group. Treatment of rats with 5 mg kg −1 Y-27632 reversed the oleic acid-induced lung damage, which was demonstrated by histopathological assessment and confirmed in Western blot experiments: ROCK-blots were more intense in the oleic acid group than in control and Y-27632 treatment reversed ROCK upregulation. In addition, malondialdehyde, myeloperoxidase, 3-nitro- l -tyrosine and nitrite/nitrate were also normalized after the administration of Y-27632 (0.5 mg kg −1 and 5 mg kg −1 ). These findings suggest that ROCK-1 and ROCK-2 are involved in oleic acid-induced lung damage in rats, and that inhibition of this enzyme by Y-27632 may have a protective effect against such damage. Consequently, Rho kinase inhibitors may be potential therapeutic agents in the treatment of acute respiratory distress syndrome (ARDS).

Objective To investigate testicular Rho-kinase levels and the effects of its inhibitor, Y-27632, on oxidative stress, spermatogenesis, and apoptosis in testicular ischemia-reperfusion rat model. Methods The study included 29 adult Wistar-Albino male rats weighing 150-200 g. The rats were divided into 3 groups. Group 1 underwent sham operation (n = 10). In group 2, left testicular torsion-detorsion was performed (n = 9). In group 3, Rho-kinase inhibitor Y-27632 (5 mg/kg) was injected intraperitoneally 30 minutes before detorsion (n = 10). Two months later, bilateral orchiectomy was performed in all the groups. Rho-kinase levels by Western blotting, apoptosis with terminal deoxynucleotidyl transferase dUTP nick end labeling method, testicular damage and spermatogenesis with modified Johnsen score, testicular total antioxidative status, and total oxidative status were measured. Results In the torsion-detorsion (T/D) group, Rho-kinase level increased significantly, compared with the sham group ( P = .025). In the Y-27632 treatment group, Johnsen scores were significantly higher, and apoptosis indexes were significantly lower, compared with the T/D group ( P = .001). Significantly higher total antioxidative status levels and lower total oxidative status levels were observed in the Y-27632 treatment group, compared with the T/D group ( P = .001 and P = .002, respectively). Conclusion Testicular ischemia-reperfusion significantly increased Rho-kinase levels in rats, and administration of Rho-kinase inhibitor, Y-27632, before detorsion might prevent ischemia-reperfusion injury.

Rho kinase (ROCK) and nitric oxide (NO) are important targets in cardiovascular diseases. Therefore, we investigated the possible influence of NO on Rho kinase (ROCK-2 isoform) expressions in cultured rat coronary microvascular endothelial cells. The cells were isolated from Wistar rats on a Langendorff system, and were incubated overnight (similar to 16 h) with an NO generator, A-23187 (10(-7) to 10(-6) M) NO donors, such as sodium nitroprusside (10(-7) to 10-6 M) glyceryl trinitrate (10(-7) to 10-6 M), 2,2'-(hydroxynitro- sohydrazono)bis-ethanimine (10(-7) to 10(-6) M), and NaNO2 (10(-4) to 10(-3) M) or a nitric oxide synthase (NOS) inhibitor, N-G-nitro-L-arginme methylester (2x10(-4) M), or two ROCK inhibitors, (-+)(R)-trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10(-5) M) and fasudil (10(-5) M) in the absence or presence of thrombin (4 U/mL). ROCK-2 and endothelial NOS (eNOS) expressions were detected by Western blotting. Moreover, nitrite/nitrate levels were detected by Griess method in the presence of the ROCK inhibitors. The NO donors and the NO generator had no significant effects on ROCK-2 expression. Y-27632 and fasudil did not alter eNOS expression and NO production. Nitrite/nitrate levels were 4.4 +/- 0.32 mu M in control and 4.0 +/- 0.93 mu M and in Y-27632 group. These results demonstrate that prolong NO donation could not suppress the expression of ROCK-2 protein, and the ROCK inhibitor did not change e-NOS expression and NO production in the cultured rat coronary microvascular endothelial cells.

Rho/Rho-kinase-mediated pathway has been involved in a variety of physiological processes, including Ca2+ sensitization, which enhances smooth muscle contraction. In this study, first of all we investigated the expression of Rho-kinase (ROCK-2) and then the role of this protein in the control of smooth muscle contraction in the isolated human gallbladder. For this purpose, we examined the effects of a selective Rho-kinase inhibitor, (+)- (R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10(-8)-3x10(-5) M) on carbachol (10(-8)-10(-4) M), cholecystokinin-8 (10(-8) M), endothelin-1 (10(-8) M), histamine (10(-5) M), neurokinin A (10(-7)-10(-6) M), 5-hydroxytryptamine (10(-6)-10(-5) M) and potassium chloride (KCl, 25-50 mM)-induced contractions as well as spontaneous contractile activity. Y-27632 (10(-5) M) significantly reduced 5-hydroxytryptamine, neurokinin A and KCl-induced contractions. Moreover, this Rho-kinase inhibitor (10(-8)-3x10(-5) M, cumulatively) relaxed the contractions produced by cholecystokinin-8, endothelin-1 and histamine in a concentration-dependent manner, being the pEC50 values for Y-27632 5.74+/-0.12, 5.33+/-0.09 and 5.95+/-0.18, respectively. Carbachol (10(-8)-10(-4) M) produced concentration-dependent contractions, which were also inhibited significantly by Y-27632. In addition, the spontaneous contractile activity was suppressed in the presence of Y-27632 (10(-6)-10(-5) M). Moreover, Western blot analysis has revealed that Rho-kinase is expressed in homogenates of the human gallbladder. Taken together, these results show that Rho-kinase is expressed in the human gallbladder, and it has an essential role in agonists and depolarization-induced contractions as well as spontaneous contractile activity.

PubMedID: 16750189 The possible antinociceptive effect of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), was investigated in mice by using the hot-plate and abdominal constriction response (writhing) tests. In addition, the expression of Rho-kinase protein (ROCK-2) was studied in the mouse brain and spinal cord by Western blotting. Male balb/c mice (n = 8, for each group) were used in the experiment. Hot-plate latency and the number of writhes were recorded in control and in Y-27632-treated (1-5 mg/kg, i.p.) groups. Y-27632 (1 mg/kg) did not affect hot-plate latency; however, it considerably diminished the number of writhes, from 89 ± 12 in control to 30 ± 6 in the mice treated with 1 mg/kg Y-27632 (P = 0.001). At a higher dose (5 mg/kg), Y-27632 prolonged the hot-plate latency from 8.7 ± 1.0 s to 14.4 ± 1.7 s (P = 0.005) and decreased the number of writhes from 80 ± 8 to 24 ± 7 (P = 0.002). Western blot analysis revealed that mouse spinal cord and brain homogenates expressed ROCK-2 protein. These results indicate that Rho-kinase may be involved in nociception and that its inhibitors, such as Y-27632, may represent a new type of antinociceptive drug. © 2006 Elsevier B.V. All rights reserved. K.B./TÜBA-GEBİP/2002-1-5 This work has been supported by the Turkish Academy of Sciences, in the framework of the Young Scientist Award Program (K.B./TÜBA-GEBİP/2002-1-5).

Background Bisphosphonates, which have been used for the treatment of the metastatic bone disease and malignant hypercalcemia due many cancers. These drugs also inhibit mevalonate pathway that results in the reduction of Ras prenylation, which eventually inhibits tumor growth. Rho/Rho kinase signaling has a fundamental role in cancer cell proliferation, migration and metastasis. In this study, we aimed to investigate the effects of y-27632 (a Rho-kinase inhibitor) and ibandronate (a bisphosphonate) and their combination on cell proliferation in prostate cell lines. Methods The adherent prostate cell line, PC-3, was grown in RPMI-1640 supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% penicillin (10.000U/ml) and streptomycin (10.000 mg/ml) in a humidified atmosphere of 5% CO2 at 37 oC for confluency. The cells were seeded in the wells of E-plates (24,000 cells/well) that allowed a real-time-monitoring of the cell index reflected cell growth and proliferation (xCELLigence, Roche). The cell index was evaluated at the different points of time (post-treatment 12, 24, 36, 48, 60 and 72 h) in the groups of, y-27632, ibandronate, the combination of these drugs and time-course control. Results Both ibandronate alone (10-100 mM) and y-27632 alone (50-100 mM) markedly decreased cell index. But the combination of ibandronate with y-27632 (in differrent concentrations but especially in higher concentrations) resulted in potentiation of cytotoxicity in prostate cell line. Conclusions Rho kinase inhibitors, bisphosphonates and their combinations may be used for the treatment of prostate cancer in the future, according to the possible beneficial results of clinical trials. Disclosure All authors have declared no conflicts of interest.

e15104 Background: Bisphosphonates, which have been used for the treatment of breast and prostate cancers (Epplen et al., 2011) inhibit mevalonate pathway that results in the reduction of Ras prenylation, which eventually inhibits tumor growth (Luckman et al., 1998). Rho/Rho kinase signaling has a fundamental role in cancer cell proliferation, migration and metastasis (Vega et al., 2008) Therefore, in this study, we aimed to investigate effects of Y-27632, which inhibits Rho-kinase, ibandronate, which inhibits farnesyl transferase and their combination on cell proliferation in prostate cell lines, PC-3. Methods: The adherent prostate cell line, PC-3 (ATCC) was grown in RPMI-1640 supplemented with 10 % fetal bovine serum, 1 % L-glutamine, 1 % penicillin (10.000U/ml) and streptomycin (10.000 mg/ml) in a humidified atmosphere of 5 % CO2 at 37oC for confluency. The cells were seeded in the wells of E-plates (24,000 cells/well) that allowed a real-time-monitoring of the cell index reflected cell growth and proliferation (xCELLigence, Roche). The cell index was evaluated at the different points of time (post-treatment 12, 24, 36, 48, 60 and 72 h) in the groups of ibandronate, Y-27632, the combination and time-course control. Results: Ibandronate (10-100 mM), Y-27632 (50-100 mM) markedly decreased cell index. The combination of Y-27632 with ibandronate resulted in potentiation of cytotoxicity in PC-3 cells. Conclusions: Rho kinase inhibitors, biphosphonates and their combination may be beneficial for the treatment of prostate cell cancers.

OBJECTIVE To investigate the expression of two isoforms of Rho-kinase (ROCK) and its functional role in the pathophysiological control of smooth muscle contraction in rabbits with unilateral ureteric obstruction (UUO). MATERIAL AND METHODS Left UUO was created in 14 rabbits and eight other rabbits (controls) had sham operations. After 2 weeks all the rabbits were killed. Ureteric strips suspended in an organ bath were used for functional studies and the effects of Y-27632, a specific inhibitor of Rho-kinase, on spontaneous contractions and electrical field stimulation (EFS; 50 V, 1 ms, 16 Hz, for 20 s), carbachol- (10−7–10−4m), phenylephrine- (10−7–10−4m) and KCl- (50 mm) induced contractions were analysed. Western blotting was used to determine expression levels of Rho-kinase protein in the ureters of UUO and control rabbits. RESULTS In the functional analysis, the contractions induced by EFS, KCl, phenylephrine and carbachol in the ureteric strips from rabbits with UUO were significantly greater than those from the control rabbits. Y-27632 considerably suppressed the ureter contractile responses in both UUO and control rabbits. Western blot analysis showed that both ROCK-1 and ROCK-2 proteins were expressed in the rabbit ureter. In accordance with the functional studies, the expression levels of both ROCK-1 and ROCK-2 were significantly greater in the ureters of UUO rabbits than in the controls. CONCLUSIONS Y-27632 suppressed ureteric contractions in the rabbits with UUO. Western blot analysis also confirmed greater expression levels of ROCK-1 and ROCK-2 in the ureters of UUO rabbits. It is important to elucidate by which mechanisms the Rho-kinase pathway affects ureteric function after obstruction.