Most cellular events are regulated by protein phosphorylation mediated by protein kinases, whose malfunction is involved in the etiology of various disorders. The elucidation of the biochemical properties of the protein phosphorylation reaction will lead not only to a better understanding of the signal transduction mechanism, but also to developing new therapeutic agents. In this review, we briefly summarize the technologies to detect or characterize protein kinases with special emphasis on recently developed and/or commercially available techniques.

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Based on the ethnomedical use of Careya arborea Roxb bark in the treatment of tumors, the present study was carried out to evaluate the anticancer potentials against Dalton’s lymphoma ascites (DLA)-induced ascitic and solid tumors. The methanol extract of its bark given orally to mice at the dose of 250 or 500 mg/kg body weight for 10 days caused significant reduction in percent increase in body weight, packed cell volume, and viable tumor cell count when compared to the mice of the DLA control group. Restoration of hematological and biochemical parameters towards normal was also observed. Histological observations of liver and kidney also indicated repair of tissue damage caused by tumor inoculation. The extract at the dose of 5 or 25 mg/kg body weight given i.p. daily for 14 days significantly reduced the solid tumor volume induced by DLA cells.

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We investigated whether leukotriene B₄ (LTB₄) and its signaling pathway play an important role in the progression of human colon cancer via a direct stimulation of cancer cell proliferation. Remarkable expression of LTB₄ receptor 1 (BLT1) in human colon cancer tissues was detected by immunohistochemistry, and Western blot analysis revealed the BLT1 expression in cultured human colon cancer cell lines, Caco2 and HT29. The 5-lipoxygenase inhibitor AA-861 and LTB₄-receptor antagonist U75302 showed negative effects on survival and proliferation of both Caco2 and HT-29 cells. The inhibition of cell proliferation is due to the apoptosis because nuclear condensation and increased annexin V expression were observed in the cells treated with AA-861 and U75302. Knockdown of BLT1 by small interfering RNA caused the suppression of BLT1 protein, resulting in the inhibition of cancer cell proliferation. Blockade of BLT1 by the receptor antagonist significantly suppresses the LTB₄-stimulated extracellular signal-regulated kinase (ERK) activation in colon cancer cells. These results indicate that the blockade of the LTB₄-signaling pathway induces apoptosis via the inhibition of ERK activation in colon cancer cells. The LTB₄-signaling pathway might be a new therapeutic target for colon cancer.

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The K⁺ channel opener nicorandil is a hybrid compound that contains nitrate in its structure. It has been reported that nicorandil can relax vascular tissue in vitro via a mechanism that involves activation of K_ATP channels and stimulation of soluble guanylyl cyclase. However, it is not known whether the increase of cGMP levels occurs through an elevation of nitric oxide (NO). The aim of the present study was to determine whether NO release was a direct effect of nicorandil. We reported here that nicorandil did not generate NO using ozone chemiluminescence detection methods in human or rat liver microsomes (P450-rich fractions) with addition of NADPH. However, nicorandil elevated cGMP levels in rat liver, aorta, and human coronary smooth muscle cells in vitro. The elevation was not inhibited by the NO trapping agent carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO). These results suggest that nicorandil elevates cGMP without NO generation.

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We examined the effect of tacrolimus (FK506), an immunosuppressive drug, on indomethacin-induced small intestinal ulceration in rats. Animals were given indomethacin (10 mg/kg, s.c.), killed 24 h later, and myeloperoxidase (MPO) activity and thiobarbituric acid reactants (TBARS) were evaluated in intestinal lesions. Tacrolimus (0.3 – 3 mg/kg) was administered p.o. twice 0.5 h before and 6 h after indomethacin injection. The expression of inducible nitric oxide synthase (iNOS) mRNA was determined by a TaqMan real-time RT-PCR, while the activity of nuclear factor (NF)-κB DNA-binding was analyzed by electrophoresis mobility shift assays (EMSA) 6 h after indomethacin treatment. Indomethacin provoked severe hemorrhagic lesions in the small intestine, mainly in the jejunum and ileum, accompanied with increases in MPO activity and TBARS. Oral administration of tacrolimus reduced the severity of indomethacin-induced intestinal lesions in a dose-dependent manner. The increases in MPO activity and TBARS were also significantly attenuated by tacrolimus. The expression of iNOS mRNA was markedly enhanced when examined 6 h after indomethacin administration, and this response was counteracted by tacrolimus. Indomethacin also activated NF-κB in a tacrolimus-preventable manner. These results suggest that tacrolimus prevents indomethacin-induced small intestinal ulceration in the rat. This effect may be due to inhibition of iNOS induction through suppression of NF-κB activation.

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Renal organic anion transporters play an important role in the handling of a number of endogenous and exogenous anionic substances in the kidney. In this study, we investigated prostaglandin E₂ (PGE₂) transport properties and intrarenal localization of mouse organic anion transporter 3 (mOat3). When expressed in Xenopus oocytes, mOat3 mediated the time- and concentration-dependent transport of PGE₂ (K_m: 1.48 μM). PGE₂ transport mediated by mOat3 was trans-stimulated by intracellular glutarate injected into the oocytes. PGE₂ efflux via mOat3 was also trans-stimulated by extracellular glutarate. Thus, mOat3 was shown to mediate the bidirectional transport of PGE₂, partly coupled to the dicarboxylate exchange mechanism. Immunohistochemical study revealed that mOat3 protein was localized at the basolateral membrane of renal proximal and distal tubules. Furthermore, diffuse expression of mOat3, including expression in the basolateral membrane in macula densa (MD) cells, was observed. These results indicate that mOat3 plays an important role as a basolateral transport pathway of PGE₂ in the distal nephron including MD cells that may constitute one of the indispensable steps for renin release and regulation of the tubuloglomerular feedback mechanism.

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We have reported that oridonin, a diterpenoid isolated from the plant Rabdosia rubescens, had apoptosis-inducing activities in many cell lines (e.g., human melanoma A375-S2, human cervical cancer HeLa, human breast adenocarcinoma MCF-7, and murine fibrosarcoma L929). In this study, we further investigated signaling events involved in oridonin-induced apoptosis in human epidermoid carcinoma A431 cells. It was found that the total tyrosine kinase activity was inhibited and the protein expressions of epidermal growth factor receptor (EGFR) and phosphorylated EGFR were decreased in oridonin-induced A431 cell apoptosis. Expression of EGFR downstream effector proteins, Grb2, Ras, Raf-1, and extracellular signal-regulated kinase (ERK), was also downregulated by oridonin. Moreover, the oridonin-induced apoptosis was augmented by the Ras inhibitor manumycin A, Raf-1 inhibitor GW5074, or ERK inhibitor PD98059, suggesting that inactivation of Ras, Raf, or ERK participates in oridonin-induced apoptosis. Taken together, oridonin-induced apoptosis in A431 cells might be through blocking EGFR and its downstream Ras/Raf/ERK signal pathway.

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To evaluate the pharmacokinetics, safety, and tolerability of solifenacin in patients with mild, moderate, or severe renal disease, eighteen patients with renal disease and six healthy volunteers received a single oral dose of solifenacin (10 mg). Pharmacokinetic parameters were assessed from blood samples drawn over a 360-h period. Safety and tolerability were also evaluated. Total mean ± S.D. exposure (ng · h/mL) to solifenacin in healthy individuals (1190 ± 403) was increased in patients with renal disease (mild: 1784 ± 792, moderate: 1559 ± 555, severe: 2530 ± 700), and elimination half-life (mean ± S.D. [h]) was prolonged (healthy: 68.2 ± 27.2, mild: 89.1 ± 34.5, moderate: 90.6 ± 27.3, severe: 111 ± 38.3). A significant correlation was found between creatinine clearance and pharmacokinetic parameters for exposure and apparent oral clearance. No deaths or serious adverse events occurred during the study. Solifenacin 10 mg was well tolerated in patients with renal disease. Solifenacin displays a higher exposure and a prolonged half-life in patients with renal impairment, especially severe. Therefore, while no special cautions are necessary for patients with mild/moderate renal impairment, patients with severe renal impairment should receive no more than 5 mg solifenacin once daily.

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The inotropic response to muscarinic receptor stimulation of isolated chick ventricular myocardium was examined at various developmental stages, and the receptor subtype involved was pharmacologically characterized. In embryonic chick ventricles, carbachol (CCh) produced positive inotropy at micromolar concentrations. In hatched chick ventricles, CCh produced negative inotropy at nanomolar concentrations. Neither positive nor negative inotropy was observed in the 19 – 21-day-old embryos. Both positive and negative inotropy were also observed with acetylcholine and oxotremoline-M. The CCh-induced positive inotropy in 7 – 9-day-old embryonic ventricles and the negative inotropy in 1 – 3-day-old hatched chick ventricles were antagonized by muscarinic receptor antagonists; pA₂ values for the positive and negative responses of pirenzepine were 7.5 and 7.2, those of AF-DX116 (11-[(2-[(diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepine-6-one) were 6.8 and 6.9, those of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were 9.0 and 8.5, and those of himbacine were 7.0 and 8.0, respectively. CCh had no effect on action potential configuration. In conclusion, the positive inotropy is most likely mediated by muscarinic M₁ receptors and the negative inotropy is mostly likely mediated by muscarinic M₄ receptors.

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In addition to their role in physiological activities, ionotropic glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) play an important role in neuronal death, especially that following ischemic insults. In this study, we examined the effect of single (SI) and twice repeated (RI)-4-vessel occlusion-ischemia on rat performance in the 8-armed radial maze test. Moreover, the effects of SI and RI on the AMPARs subunits glutamate receptor (GluR) 1 and GluR2 flip and flop variants composition in the CA1 subregion of the hippocampus were investigated using RT-PCR, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and expressed as their ratios to the latter. The results showed that SI and RI impaired the maze performance by decreasing correct choices and increasing the error choices, but RI increased error choices to a greater extent than the SI. The SI reduced only GluR1 flip/GAPDH on day 1. The SI did not alter ratios of GluR2 variants to those of GluR1. On the other hand, the RI decreased GluR2 flip and flop variants after 1 and 3 days, respectively, whereas after 7 days, it increased the flip variant of both GluR1 and GluR2. Moreover, the RI reduced ratios of GluR2 variants to those of GluR1. These results reveal the differential effects of the SI and RI on memory and expression of the AMPARs subunits GluR1 and GluR2 and their flip and flop variants in the CA1.

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One possible mechanism underlying inflammation-induced sensitization of the primary afferent neuron is the upregulation of tetrodotoxin-resistant (TTX-R) Na⁺ current by inflammatory mediators such as prostaglandins. This notion is based on reports that showed an augmentation of TTX-R Na⁺ current following an application of prostaglandin E₂ (PGE₂) in dorsal root ganglion (DRG) neurons. However, no information was available on the properties of the novel type of TTX-R Na⁺ channel, Na_V1.9, at times when these reports were published. Hence, the contribution of Na_V1.9 to the PGE₂-induced upregulation of TTX-R Na⁺ current remains to be elucidated. To further examine the modulation of TTX-R Na⁺ current by PGE₂, we recorded two components of TTX-R Na⁺ current in isolation from small (<25 μm in diameter) DRG neurons using wild-type and Na_V1.8 knock-out mice. Unexpectedly, neither the component mediated by Na_V1.8 nor the persistent component mediated by Na_V1.9 was affected by PGE₂ (1 and 10 μM). Our results raise a question regarding the well-known modulatory role of PGE₂ on TTX-R Na⁺ current in inflammatory hyperalgesia.

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We established an experimental system for measuring blood flow in the rat fundus and examined whether intravenously administered vasodilatory prostaglandins (PGE₁, PGE₂, and PGI₂), 8-(4-chlorophenylthio)-cAMP (a cAMP analogue), and nicardipine (a Ca²⁺-channel blocker) increase fundus blood flow (FBF). Under artificial ventilation, rats were injected with tetrodotoxin (50 μg/kg, i.v.) to eliminate any nerve activity and prevent movement of the eye. After tetrodotoxin, the rats were infused with norepinephrine (0.3 – 0.5 μg · kg⁻¹ · min⁻¹) and epinephrine (2.7 – 4.5 μg · kg⁻¹ · min⁻¹) simultaneously to maintain adequate systemic circulation. We found that intravenous infusion of PGE₁ (2 – 10 μg · kg⁻¹ · min⁻¹), PGE₂ (3 – 30 μg · kg⁻¹ · min⁻¹), and PGI₂ (1 – 10 μg · kg⁻¹ · min⁻¹) increased the FBF in a dose-dependent manner. The vasodilatory PGs decreased arterial pressure, whereas they did not affect heart rate. Like vasodilatory PGs, 8-(4-chlorophenylthio)-cAMP (30 μmol/kg, i.v.) increased FBF and decreased arterial pressure. While infusion of nicardipine (0.3 – 3 μg · kg⁻¹ · min⁻¹) produced comparable depressor responses with those to vasodilatory PGs and the cAMP analogue, it did not increase FBF. These results suggest that vasodilatory PGs and cAMP act more selectively than Ca²⁺-channel blockers on retinal/choroidal blood vessels. Therefore, the vasodilatory PGs might be considered to be possible candidates for the therapeutics to treat disorders of retinal/choroidal circulation.

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Quantification of arthritis is helpful for investigating pain mechanisms of arthritis and for developing new drugs. We assessed and identified a feasible parameter for quantification of rat arthritis using a novel gait analyzing system. Knee-joint injection with small doses of λ-carrageenan decreased swing time ratio (STR, swing time of the non-treated hindlimb/swing time of the λ-carrageenan-injected hindlimb) in a dose-dependent manner. Intraperitoneal treatment with indomethacin restored the decreased STR dose-dependently. The arthritis could not be accurately quantified by swing time and swelling, common indices of arthritis. These results show that STR is a sensitive, reliable parameter for quantification of arthritis.

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The effects of edaravone, a curative agent for acute brain infarction, on singlet oxygen (¹O₂) released from activated human neutrophils were examined, and the effects were compared to those of histidine, a ¹O₂ singlet oxygen scavenger. The neutrophils, stimulated with opsonized zymosan, released ¹O₂ that was detected by chemiluminescence using a ¹O₂ specific probe, trans-1-(2'-methoxyvinyl)pyrene. Edaravone dose-dependently suppressed the ¹O₂ release with an IC₅₀ of approximately 0.3 μM, while the IC₅₀ of histidine was approximately 1 mM. This ¹O₂ scavenging activity of edaravone might be involved in its curative effects on acute brain infarction.

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We examined the effects of methyl-β-cyclodextrin (MβCD) on Ca²⁺-induced Ca²⁺ release (CICR) in smooth muscle cells (SMCs) of mouse urinary bladder (UB). Short depolarization of UBSMCs under voltage-clamp elicited several local Ca²⁺ transients (Ca²⁺ hot spots) via CICR within 20 ms in discrete sub-sarcolemmal areas. Then, the Ca²⁺ wave spread to whole areas. The pretreatment with 10 mM MβCD significantly attenuated Ca²⁺ hot spots in UBSMCs and reduced contraction by single direct electrical pulse stimulation in UBSM strips. MβCD may prevent CICR by attenuating the coupling between voltage-dependent Ca²⁺ channels and ryanodine receptors in Ca²⁺ hot spot areas.

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