A novel soluble guanylate cyclase activator with reduced risk of hypotension by short-acting vasodilation.

Cinaciguat, a soluble guanylate cyclase (sGC) activator, was under clinical development for use in acute decompensated heart failure (ADHF), but was discontinued due to occurrence of hypotension. We hypothesized that short-term activation of sGC in ADHF patients would exert a vasodilative effect without hypotension irrespective of disease state, using a novel short-acting sGC activator, TY-55002. The objective of this study was to investigate the vasodilation and hemodynamic effects of TY-55002 in comparison with those of cinaciguat. TY-55002 and cinaciguat activated both normal and heme-oxidized sGC in a dose-dependent manner and caused rapid relaxation of phenylephrine-contracted rat aorta. However, TY-55002 had a milder effect than cinaciguat in enhancing the dose-activity response between normal and oxidized sGC. Therefore, we suggest that the pharmacological effect of TY-55002 is less subject than cinaciguat to oxidative stress associated with complications such as cardiovascular disease or diabetes. In normal dogs, the effects of intravenous TY-55002 or cinaciguat on blood pressure were evaluated in conjunction with the plasma concentrations of the compounds, and pharmacokinetic (PK)-pharmacodynamic (PD) analyses were carried out. The plasma-to-effect-site transfer rate constant (Ke0) for TY-55002 was three times greater than for cinaciguat. On the other hand, there was a small difference in blood half-life (T1/2) between the compounds. It is possible that the rapid fall in blood pressure after the initial administration of TY-55002 and the quick recovery after cessation were due to the pharmacodynamic property of the compound. In heart failure-model dogs, TY-55002 and cinaciguat improved the condition to the same degree, and the short-term action of TY-55002 was replicated. In conclusion, TY-55002 is a novel short-acting sGC activator, which offers the possibility of easy dose management without excessive hypotension. It therefore holds potential to serve as an innovative drug in the pharmacotherapy of ADHF.

The cardioprotective effects of novel Na+/H+ exchanger inhibitor TY-51924 on ischemia/reperfusion injury.

The inhibitory effects of sodium 3-guanidinocarbonyl-2-methyl-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-9-ylmethyl sulfate monoethanolate (TY-51924) are selective for Na(+)/H(+) exchanger (NHE)-1 in PS120 cells expressing human NHE isoforms assayed by NH(4)Cl prepulse technique. The median inhibitory concentrations (micromolar) of TY-51924 were 0.095 ± 0.008 (NHE-1), 0.621 ± 0.093 (NHE-2), and >100 (NHE-3). In anesthetized dogs subjected to 90 minutes ischemia/300 minutes reperfusion, intravenous bolus TY-51924 at 5 and 10 mg/kg administered 5 minutes before reperfusion reduced infarct size. The infarct size reduction ratios of TY-51924 at 5 and 10 mg/kg versus vehicle were 32.8% and 52.4%, respectively. But TY-51924 at 10 mg/kg administered 10 minutes after reperfusion did not reduce infarct size. In 2-step intravenous infusion initiated 15 minutes before reperfusion, TY-51924 at low dose (3.8 mg/kg per 5 minutes, then 6.2 mg/kg per 20 minutes) and at high dose (7.6 mg/kg per 5 minutes, then 12.4 mg/kg per 20 minutes) reduced infarct size. The infarct size reduction ratios of TY-51924 at 10 and 20 mg/kg versus vehicle were 39.2% and 51.7%, respectively; plasma drug concentrations at reperfusion were 16.8 and 38.8 µg/mL, respectively. This indicates that maintaining a plasma drug concentration of >20 µg/mL at reperfusion enables TY-51924 to reduce infarct size by inhibiting the NHE, which is activated during the early period of reperfusion.

R-Ras regulates exocytosis by Rgl2/Rlf-mediated activation of RalA on endosomes.

R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis.

GTP hydrolysis by the Rho family GTPase TC10 promotes exocytic vesicle fusion.

TC10, a Rho family GTPase, has been shown to play an important role in the exocytosis of GLUT4 and other proteins, primarily by tethering the vesicles at the plasma membrane. Using a newly developed probe based on fluorescence resonance energy transfer, we found that TC10 activity at tethered vesicles dropped immediately before vesicle fusion in HeLa cells stimulated with epidermal growth factor (EGF), suggesting that GTP hydrolysis by TC10 is a critical step in vesicle fusion. In support of this model, a GTPase-deficient TC10 mutant potently inhibited EGF-induced vesicular fusion in HeLa cells and depolarization-induced neuronal secretion. Furthermore, we found that GTP hydrolysis by TC10 in the vicinity of the plasma membrane was dependent on Rac and the redox-regulated Rho GAP, p190RhoGAP-A. We propose that an EGF-stimulated GAP accelerates GTP hydrolysis of TC10, thereby promoting vesicle fusion.

RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney cells.

RalA, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using fluorescence resonance energy transfer-based probes for RalA activity, we found that the EGF-induced RalA activation in Cos7 cells was restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurred more diffusely at the plasma membrane. This EGF-induced RalA activation was not observed when lamellipodial protrusion was suppressed by a dominant negative mutant of Rac1, a GTPase-activating protein for Cdc42, inhibitors of phosphatidylinositol 3-kinase, or inhibitors of actin polymerization. On the other hand, EGF-induced lamellipodial protrusion was inhibited by microinjection of the RalA-binding domains of RalBP1 and Sec5. Furthermore, we found that RalA activity was high at the lamellipodia of migrating Madin-Darby canine kidney cells and that the migration of Madin-Darby canine kidney cells was perturbed by the microinjection of RalBP1-RalA-binding domain. Thus, RalA activation is required for the induction of lamellipodia, and conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. Our observation also demonstrates that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral guanine nucleotide exchange factors.

Cloning, expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis.

Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent K(m) values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni(2+)-affinity column were 24.1, 6.8, 2.3, and 11.5 microM, respectively. The enzyme shows optimum activity at approximately 40 degrees C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.