OBJECTIVE: We examined the vasoconstricting poperties of endothelin (ET) on isolated arteries from pregnant as well as non-pregnant uterus. METHODS: Arteries of the uterus were obtained from both hysterectomized uterus and during pregnany hysterectomy for control group and cesarean section for pregnant group. Rings of uterine artery were suspended on muscle chambers at their optimal length for generating tension and contractile properties were examined. RESULTS: ET-1 and ET-2 induced concentration-dependent constriction of both isolated arterial strips from non-pregnant and pregnant uterus. The contraction to ET-1 and ET-2 were more enhanced in full-term pregnancy. Furthermore, in pregnant group, sarafotoxin S6c and IRL 1620, ET. agonists, induced a dose-dependent contraction, which was not shown in those from non-pregnant human. Pretreatment of human uterine arterial strips from pregnant uterus with BQ610, an ET. antagonist, for 10 min resulted in a dose-related rightward shift of ET-1 response curve with diminution of maximal response. Schild plot analysis yielded a pA value of 7.29 with a slope of 0.98. However, BQ788, an ET antagonist, did not produce any rightward shift. The contraction to lower concentration (10-8~3*10-7 M) of sarafotoxin S6c was not affected by BQ788, whereas that to higher concentration (10-s-8*10-7 M) was marked diminished. However, BQ610 did not exnt any efFect on sarafotoxin S6c-induced contraction in arterial staips from pregnant uterus. When the bath solution was replaced with Ca-free physiological salt solution (PSS) containing 1 mM EGTA for 10 min prior to adding sarafotoxin S6c, sarafotoxin S6c-induced contraction was completely abolished. Sarafotoxin S6c (10 nM)-induced contraction was prefetentially blocked by a protein kinase C antagonist, H-7, whereas it was less sensitive to a calmodulin antagonist, calmidazolium, CONCLUSION: Based on above results, we concluded that ET plays an important role in regulating uterine blood flow through the activation of ETa and ETB receptors. Furthermote, ETB receptors may predominantly contribute to the modulation of human uterine circulation in full-term pregnancy.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; Arteries* ; Baths ; Calmodulin ; Cesarean Section ; Constriction ; Egtazic Acid ; Endothelin-2 ; Endothelins* ; Female ; Humans* ; Hysterectomy ; Pregnancy ; Protein Kinase C ; Receptors, Endothelin ; Uterine Artery ; Uterus*

In rapidly growing tumors, hypoxia commonly develops due to the imbalance between O2 consumption and supply. Hypoxia Inducible Factor (HIF)-1alpha is a transcription factor responsible for tumor growth and angiogenesis in the hypoxic microenvironment; thus, its inhibition is regarded as a promising strategy for cancer therapy. Given that CamKII or PARP inhibitors are emerging anticancer agents, we investigated if they have the potential to be developed as new HIF-1alpha-targeting drugs. When treating various cancer cells with the inhibitors, we found that a CamKII inhibitor, KN-62, effectively suppressed HIF-1alpha specifically in hepatoma cells. To examine the effect of KN-62 on HIF-1alpha-driven gene expression, we analyzed the EPO-enhancer reporter activity and mRNA levels of HIF-1alpha downstream genes, such as EPO, LOX and CA9. Both the reporter activity and the mRNA expression were repressed by KN-62. We also found that KN-62 suppressed HIF-1alpha by impairing synthesis of HIF-1alpha protein. Based on these results, we propose that KN-62 is a candidate as a HIF-1alpha-targeting anticancer agent.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; Anoxia ; Antineoplastic Agents ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Carcinoma, Hepatocellular ; Gene Expression ; RNA, Messenger ; Transcription Factors

It has been suggested that Ca2+ sensitization mechanisms might contribute to myogenic tone, however, specific mechanisms have not yet been fully identified. Therefore, we investigated the role of protein kinase C (PKC)- or RhoA-induced Ca2+ sensitization in myogenic tone of the rabbit basilar vessel. Myogenic tone was developed by stretch of rabbit basilar artery. Fura-2 Ca2+ signals, contractile responses, PKC immunoblots, translocation of PKC and RhoA, and phosphorylation of myosin light chains were measured. Stretch of the resting vessel evoked a myogenic contraction and an increase in the intracellular Ca2+ concentration ([Ca2+]i) only in the presence of extracellular Ca2+. Stretch evoked greater contraction than high K+ at a given [Ca2+]i. The stretch-induced increase in [Ca2+]i and contractile force were inhibited by treatment of the tissue with nifedipine, a blocker of voltage-dependent Ca2+ channel, but not with gadolinium, a blocker of stretch-activated cation channels. The PKC inhibitors, H-7 and calphostin C, and a RhoA-activated protein kinase (ROK) inhibitor, Y-27632, inhibited the stretch-induced myogenic tone without changing [Ca2+]i. Immunoblotting using isoform-specific antibodies showed the presence of PKCalpha and PKCepsilon in the rabbit basilar artery. PKCalpha, but not PKCepsilon, and RhoA were translocated from the cytosol to the cell membrane by stretch. Phosphorylation of the myosin light chains was increased by stretch and the increased phosphorylation was blocked by treatment of the tissue with H-7 and Y-27632, respectively. Our results are consistent with important roles for PKC and RhoA in the generation of myogenic tone. Furthermore, enhanced phosphorylation of the myosin light chains by activation of PKCalpha and/or RhoA may be key mechanisms for the Ca2+ sensitization associated with myogenic tone in basilar vessels.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; Antibodies ; Basilar Artery ; Calcium ; Cell Membrane ; Cytosol ; Fura-2 ; Gadolinium ; Immunoblotting ; Myosin Light Chains ; Nifedipine ; Phosphorylation ; Protein Kinase C ; Protein Kinases

Impaired endothelium dependent relaxation occurs in diabetic rabbit corpus cavernosum and normal corpus cavernosum exposed to elevated glucose. Elevation of glucose can change the activities of two key enzymes, protein kinase C (PKC) and Na+-K+ ATPase. This study addresses the question of whether impaired endothelium dependent relaxation in isolated corpus cavernosum from normal rabbit exposed to elevated glucose is related to PKC and Na+-K+ ATPase activities and, if so, whether it is associated with altered ouabain sensitive 86-Rb uptake, an index of Na+-K+ ATPase activity, and contractile response of corpus cavernosum tissue to ouabain. Corpus cavernosal tissue suspended for measurement of isometric tension were incubated for 6 hours in control (5.5mM) or elevated glucose (44mM) to mimic euglycemic and hyperglycemic conditions. Relaxations of corpus cavernosum tissue in response to the endothelium-dependent vasodilator acetylcholine (ACh)were unaffected in control groups while significantly inhibited in the elevated glucose group. Relaxations of corporeal tissue to endothelium-independent vasodilators, sodium nitroprusside (SNP) were similar in the control and elevated glucose groups. Corporeal tissue treated with 4-phorbol 12-myristate 13-acetate (PMA), a PKC activator, showed decreased relaxations to ACh, similar to normal corporeal tissue exposed to elevated glucose. Relaxations in response to SNP were unaffected by treatment with PMA or exposure to elevated glucose. 1(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), PKC inhibitor, restored the abnormal Ach-induced relaxation in corporeal tissue exposed to elevated glucose. The contractions caused by ouabain, Na+-K+ ATPase inhibitor, were smaller in elevated glucose groups than control and elevated glucose groups treated with H-7. Ouabain sensitive 86-Rb uptake of elevated glucose groups was significantly less than that of control groups but ouabain sensitive 86-Rb uptake of elevated glucose groups treated with H-7 was similar to those of control groups. These results suggest that activation in PKC activity and inhibition in Na+-K+ ATPase activity caused by elevated glucose contribute to impaired endothelium dependent relaxation in corpus cavernosum smooth muscle.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; Acetylcholine ; Adenosine Triphosphatases* ; Endothelium ; Glucose* ; Muscle, Smooth ; Nitroprusside ; Ouabain ; Protein Kinase C* ; Protein Kinases* ; Relaxation* ; Vasodilator Agents

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Hypertension, Pulmonary ; drug therapy ; Hypoxia ; pathology ; Protein Kinase Inhibitors ; pharmacology ; Rats ; rho-Associated Kinases ; antagonists & inhibitors

The study aims to identify the related substances in fasudil hydrochloride by hyphenated techniques. A WondaSil C18 (250 mm x 4.6 mm, 5 microm) column was used for the separation of the related substances with a mixture of methanol and ammonium acetate buffer solution as the mobile phase by gradient elution. The structures of the related substances were speculated by electrospray positive ionization LC-TOF/MS accurate ion mass and MS/MS determination and elucidation, and verified further through synthesis and spectroscopic analysis. Fasudil hydrochloride and the related substances were separated under the established HPLC condition. Three related substances in fasudil hydrochloride were characterized by hyphenated techniques. The hyphenated LC-MS method is useful for the identification of related substances in fasudil hydrochloride and the results obtained are valuable for its manufacturing process and quality control.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; chemistry ; Calcium Channel Blockers ; chemistry ; Chromatography, Liquid ; Drug Contamination ; Quality Control ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry ; Vasodilator Agents ; chemistry

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Cardiomyopathy, Hypertrophic ; drug therapy ; metabolism ; Connexin 43 ; metabolism ; Disease Models, Animal ; Male ; Myocardium ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effect of fasudil, an inhibitor of Rho kinase, on the erectile function of hypertensive rats and its action mechanism.

METHODSTwenty 12-week-old healthy male Sprague-Dawley rats were randomly divided into groups A (control), B (hypertension) and C (fasudil treatment). After establishment of the hypertension model, group C received intraperitoneal injection of fasudil at 30 mg/(kg x d), while A and B normal saline only. At 10 weeks after surgery, we measured the corpus cavernosum pressure/mean carotid arterial pressure (ICPmax / MAP), and the expression levels of ROCK1 and ROCK2 proteins in the corpus cavernosum of the rats by Western-blot.

RESULTSThe systolic blood pressure (mmHg) and the expressions of ROCK1 and ROCK2 proteins were significantly increased in group B (190.39 +/- 5.07, 0.048 +/- 0.002 and 0.143 +/- 0.011) as compared with A (124.81 +/- 4.01, 0.036 +/- 0.001 and 0.101 +/- 0.011) (P<0.05), but markedly decreased in group C (182.03 +/- 4.32, 0.044 +/- 0.001 and 0.126 +/- 0.007) in comparison with B (P<0.05). ICPmax /MAP was significantly lower in group B (36.82 +/- 5.47) than in A (59.99 +/- 5.69) (P<0.05), but remarkably higher in group C (51.1 +/- 5.63) than in B (P<0.05).

CONCLUSIONFasudil can improve erectile function in hypertensive rats by inhibiting the expression of RhoA / Rho kinase signaling and its possible attenuating effect on hypertension.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; therapeutic use ; Animals ; Hypertension ; physiopathology ; Male ; Penile Erection ; drug effects ; Rats ; Rats, Sprague-Dawley ; Vasodilator Agents ; pharmacology ; therapeutic use

OBJECTIVETo study the expression of Rho/Rho kinase of cardiac muscle in heart failure rats caused by pressure overload and the effects of fasudil on heart failure.

METHODSThe heart failure models were successfully induced by coarctation of ascending aorta after 20 weeks in this study. Thirty female Wistar operated rats were divided randomly into three groups (n = 10) for 4 week treatment. (1) Sham operation group: normal saline, 0.1 ml, i.p,Bid. (2) Heart failure group: normal saline, 0.1 ml,i.p,Bid. (3) Fasudil group: fasudil 5 mg/kg, i.p, Bid. The hemodynamic parameters, the ratio of LV weight to body weight, the expressions of RhoA and Rho kinase mRNA, and the concentration of calcium ion same as [Ca(2+)](i) were investigated in the three groups.

RESULTSHemodynamic parameters were significantly changed in heart failure group than those in sham operation group, such as left ventricular diastolic end pressure increased [(13.00 +/- 0.30) mm Hg vs (3.78 +/- 0.31) mm Hg, P < 0.01], left ventricular systolic pressure decreased [(97.20 +/- 7.21) mm Hg vs (129.45 +/- 7.52) mm Hg, P < 0.01]. Those results could be significantly changed by use of fasudil, P < 0.01. The ratio of LV weight to body weight was significantly increased in heart failure group than that in sham operation group [(4.77 +/- 0.08) mg/g vs (2.51 +/- 0.12) mg/g, P < 0.01]. Fasudil could significantly decrease the ratio of LV weight to body weight compared with that in heart failure group [(4.05 +/- 0.08) mg/g vs (4.77 +/- 0.08) mg/g, P < 0.01]. Cardiac muscle RhoA, Rho kinase mRNA level and [Ca(2+)](i) were higher in heart failure group than those in sham operation group [Ca(2+)](i) (475.93 +/- 28.22) nmol/L vs (79.25 +/- 3.33) nmol/L, P < 0.01. Compared with those in heart failure group, the expressions of RhoA, Rho kinase mRNA level decreased significantly, P < 0.01, and the levels of cardiomyocyte [Ca(2+)](i) had no change in fasudil group [(462.78 +/- 16.72) nmol/L vs (475.93 +/- 28.22) nmol/L, P > 0.05].

CONCLUSIONSThese results indicated that heart failure was probably related to activating of RhoA, Rho kinase. Fasudil may contribute to the observed beneficial effects on heart failure such as the decrease of RhoA, Rho kinase mRNA expression and not increase of [Ca(2+)](i) level. Rho/Rho kinase may be a novel, potent signaling of heart failure.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Female ; Heart Failure ; metabolism ; physiopathology ; Myocardium ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Rats ; Rats, Wistar ; rho-Associated Kinases ; metabolism

To investigate the mechanism of smooth muscle contraction induced by emptying of intracellular Ca2+ stores, we measured isometric contraction and 45Ca2+ influx. CaCl2 increased Ca2+ store emptying- induced contraction in dose-dependent manner, but phospholipase C activity was not affected by the Ca2+ store emptying-induced contraction. The contraction was inhibited by voltage-dependent Ca2+ channel antagonists dose dependently, but not by TMB-8 (intracellular Ca2+ release blocker). Both PKC inhibitors (H-7 and staurosporine) and tyrosine kinase inhibitors (genistein and methyl 2,5-dihydroxycinnamic acid) significantly inhibited the contraction, but calmodulin antagonists (W-7 and trifluoperazine) had no inhibitory effect on the contraction. The combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were greater than that of each one alone. In Ca2+ store-emptied condition, 45Ca2+ influx was significantly inhibited by verapamil, H-7 or genistein but not by trifluoperazine. However combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were not observed. Therefore, this kinase pathway may modulate the sensitivity of contractile protein. These results suggest that contraction induced by emptying of intracellular Ca2+ stores was mediated by influx of extracellular Ca2+ through voltage-dependent Ca2+ channel, also protein kinase C and/or tyrosine kinase pathway modulates the Ca2+ sensitivity of contractile protein.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; Animals ; Calmodulin ; Cats* ; Genistein ; Isometric Contraction ; Muscle, Smooth* ; Phosphotransferases ; Protein Kinase C ; Protein Kinase Inhibitors ; Protein-Tyrosine Kinases ; Trifluoperazine ; Type C Phospholipases ; Verapamil