The relationship between flow-mediated brachial artery vasodilation and coronary vasomotor responses to bradykinin: comparison with those to acetylcholine

Flow-mediated dilation (FMD) of brachial artery provides a noninvasive assessment of coronary endothelial dysfunction. Acetylcholine (ACh) has been used as an agent for estimating coronary endothelial function. In contrast to ACh, there is no evidence for a relationship between FMD and coronary vasodilation to bradykinin (BK). The aim of this study was to compare the flow-mediated vasodilation of brachial artery with coronary vasomotor responses to intracoronary ACh or BK in patients with an angiographically normal left anterior descending coronary artery. Ninety-one patients underwent the cardiac catheterization examination with coronary endothelial function testing and the brachial ultrasound study. BK (0.2, 0.6, 2.0 microg/min) and ACh (3, 10, 30 microg/min) were administered into the left coronary artery in a stepwise manner. Coronary blood flow was evaluated by the Doppler flow velocity measurement. Coronary diameters were measured by the quantitative coronary angiography. The assessment of endothelial function in the brachial artery was made in response to reactive hyperemia with high-resolution ultrasound. Bradykinin induced dose-dependent increases in epicardial coronary diameter and blood flow. There was a significant positive correlation between FMD- and BK-induced vasodilations of epicardial coronary arteries (0.2 microg/min: r = 0.30; 0.6 microg/min: r = 0.42; 2.0 microg/min: r = 0.44, P < 0.01, respectively) and resistance coronary arteries (0.2 microg/min: r = 0.40; 0.6 microg/min: r = 0.56; 2.0 microg/min: r = 0.59, P < 0.0001, respectively). FMD correlated with ACh-induced vasomotions of resistance but not epicardial coronary arteries. No correlation was seen between nitroglycerin-induced brachial artery vasodilation and BK-induced coronary vasodilation. The endothelial dysfunction of peripheral arteries correlated well with that of the coronary arteries especially vasomotor responses to BK.     

Bepridil block of recombinant human cardiac IKs current shows a time-dependent unblock

Whole-cell patch-clamp techniques were employed to examine the effects of bepridil, a Ca2+ channel blocker with Vaughan Williams class III action, on a slow component of cardiac delayed rectifier K+ current (IKs), which was reconstituted in HEK293 cells by transfecting KCNQ1 and KCNE1. Micromolar bepridil inhibited tail currents carried by KCNQ1/KCNE1 channels in a concentration-dependent manner (IC50 = 5.3 +/- 0.7 microM at -40 mV from 1000 milliseconds test pulse). When the effect of the drug was examined with a short test pulse protocol (250 milliseconds), IC50 became two-fold smaller than that measured with 1000 milliseconds test pulse (2.5 +/- 0.8 microM). The envelope-of-tails protocol was used to assess how the duration of depolarizing pulse affects the drug action on the outward KCNQ1/KCNE1 channel current. The drug significantly inhibited tail currents more potently during shorter pulses (<600 milliseconds). Bepridil's block was therefore time dependent, and its binding affinity to the channel was greater in the closed state channel, as evidenced by unblocking during prolonged depolarization. These properties of channel blockade appear to underscore the mechanism of bepridil's effect on IKs current.     

Effect of two different groups of Chinese medicines on nitric oxide production by mouse macrophage-like cells

Seven Chinese medicines were investigated for their ability to modify nitric oxide (NO) production by unstimulated and lipopolysaccharide (LPS)-stimulated mouse macrophage-like Raw 264.7 cells, in comparison with their radical intensity and scavenging activity. LPS significantly stimulated the NO production by Raw 264.7 cells. Three Chinese medicines, Shosaiko-to, Hange-shashin-to and Sairei-to (tentatively classified as Group I), significantly reduced the extracellular concentration of NO in the LPS-stimulated cells, slightly below their cytotoxic concentrations. On the other hand, another four Chinese medicines, Byakko-ka-ninjin-to, Hochu-ekki-to, Juzen-taiho-to and Ninjin-yoei-to (tentatively classified as Group II), showed similar effects, but required higher concentrations due to the co-existence of both the inhibitors and stimulators for NO production by activated macrophages. Western blot analysis demonstrated that LPS stimulated the expression of inducible NO synthase (iNOS) at both protein and mRNA levels, and that Sairei-to reduced the LPS-induced iNOS expression more potently than did Juzen-taiho-to. ESR spectroscopy shows that Group I medicines generally produced higher amounts of radicals under alkaline condition, and scavenged superoxide (produced by hypoxanthine-xanthine oxidase reaction) and NO (produced by NOC-7, NO generator) more potently than Group II medicines. These data support the classification of Chinese medicines into two groups: Group I and Group II. The net inhibition of NO production by Group I medicines may be the summation of the radical scavenging activity and the inhibition of iNOS expression due to higher cytotoxicity. Group II medicines showed lower cytotoxicity, lower radical intensity, lower radical scavenging activity, but higher stimulation activity for NO production by macrophages than Group I, suggesting their possible application for immunopotentiation.     

Implication of CDK inhibitors p21 and p27 in the differentiation of HL-60 cells

All-trans retinoic acid (ATRA) differentiates HL-60 cells into granulocyte-like cells and cellular proliferation is repressed markedly along with the morphological and physiological changes specific for cellular differentiation. To elucidate the implication of cyclin-dependent kinase (CDK) inhibitors during differentiation, we examined the expression of CDK inhibitors during the differentiation of HL-60 cells. The expression of p21 and p27 among the CDK inhibitors we examined increased during the differentiation induced with ATRA. Then, we established stable transformants of HL-60 cells expressing antisense RNA for p21 and p27 and examined the ability of these cells to differentiate into granulocyte-like cells. The extents of fully differentiated HL-60 cells transfected with genes for antisense RNA of p21 and p27 were only 53% and 60%, respectively, whereas 90% of the parental HL-60 cells differentiated by the ATRA treatment. These results suggest that increased expression of CDK inhibitors, p21 and p27, is necessary for the differentiation of HL-60 cells induced with ATRA.     

Effects of synthetic sphingosine-1-phosphate analogs on arachidonic acid metabolism and cell death

Sphingolipid metabolites such as sphingosine regulate cell functions including cell death and arachidonic acid (AA) metabolism. D-erythro-C18-Sphingosine-1-phosphate (D-e-S1P), a sphingolipid metabolite, acts as an intracellular messenger in addition to being an endogenous ligand of some cell surface receptors. The development of S1P analogs may be useful for studying and/or regulating S1P-mediated cellular responses. In the present study, we found that several synthetic S1P analogs at pharmacological concentrations stimulated AA metabolism and cell death in PC12 cells. D-erythro-N,O,O-Trimethyl-C18-S1P (D-e-TM-S1P), L-threo-O,O-dimethyl-C18-S1P (L-t-DM-S1P) and L-threo-O,O-dimethyl-3O-benzyl-C18-S1P (L-t-DMBn-S1P) at 100 microM stimulated [(3)H]AA release from the prelabeled PC12 cells. L-t-DMBn-S1P at 20 microM increased prostanoid formation in PC12 cells. L-t-DMBn-S1P-induced AA release was inhibited by D-e-sphingosine, but not by the tested PLA(2) inhibitors. L-t-DMBn-S1P did not stimulate the activity of cytosolic phospholipase A(2alpha) (cPLA(2alpha)) in vitro and the translocation of cPLA(2alpha) in the cells, and caused AA release from the cells lacking cPLA(2alpha). These findings suggest that L-t-DMBn-S1P stimulated AA release in a cPLA(2alpha)-independent manner. In contrast, D-e-S1P and D-erythro-N-monomethyl-C18-S1P caused cell death without AA release in PC12 cells, and the effects of D-e-TM-S1P, L-t-DM-S1P and L-t-DMBn-S1P on cell death were limited. Synthetic S1P analogs may be useful tools for studying AA metabolism and cell death in cells.     

Cancer chemopreventive effects of polyunsaturated fatty acids

The inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), in Raji cells as a primary screening test for anti-tumor promoters, for 22 fatty acids (as free and esterified forms), including 10 di- and polyunsaturated acids, and the inhibitory effects on activation of (+/-)-(E)-methtyl-2-[(E)-hydroxy-imino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor, as a primary screening test for anti-tumor initiators, for 17 fatty acids (as methyl ester forms), were evaluated. Among the fatty acids tested, three n-3 polyunsaturated acids, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), exhibited potent inhibitory effects both on EBV-EA and NOR 1 activation. Furthermore, DHA methyl ester exhibited remarkable anti-tumor-promoting activity on an in vivo two-stage carcinogenesis test of mouse tumor using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter.     

Apical/Basolateral Surface Expression of Drug Transporters and its Role in Vectorial Drug Transport

It is well known that transporter proteins play a key role in governing drug absorption, distribution, and elimination in the body, and, accordingly, they are now considered as causes of drug–drug interactions and interindividual differences in pharmacokinetic profiles. Polarized tissues directly involved in drug disposition (intestine, kidney, and liver) and restricted distribution to naive sanctuaries (blood–tissue barriers) asymmetrically express a variety of drug transporters on the apical and basolateral sides, resulting in vectorial drug transport. For example, the organic anion transporting polypeptide (OATP) family on the sinusoidal (basolateral) membrane and multidrug resistance-associated protein 2 (MRP2/ABCC2) on the apical bile canalicular membrane of hepatocytes take up and excrete organic anionic compounds from blood to bile. Such vectorial transcellular transport is fundamentally attributable to the asymmetrical distribution of transporter molecules in polarized cells. Besides the apical/basolateral sorting direction, distribution of the transporter protein between the membrane surface (active site) and the intracellular fraction (inactive site) is of practical importance for the quantitative evaluation of drug transport processes. The most characterized drug transporter associated with this issue is MRP2 on the hepatocyte canalicular (apical) membrane, and it is linked to a genetic disease. Dubin–Johnson syndrome is sometimes caused by impaired canalicular surface expression of MRP2 by a single amino acid substitution. Moreover, single nucleotide polymorphisms in OATP-C/SLC21A6 (SLCO1B1) also affect membrane surface expression, and actually lead to the altered pharmacokinetic profile of pravastatin in healthy subjects. In this review article, the asymmetrical transporter distribution and altered surface expression in polarized tissues are discussed.     

Inhibitory effects of fungal bis(naphtho-gamma-pyrone) derivatives on nitric oxide production by a murine macrophage-like cell line, RAW 264.7, activated by lipopolysaccharide and interferon-gamma

Inhibitory effects of six fungal bis(naphtho-gamma-pyrone) derivatives on nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide and interferon-gamma were examined. Among these derivatives, chaetochromin (4) (IC(50): 0.8 microM), cephalochromin (1) (IC(50) 1.5 microM), and dihydroisoustilaginoidin A (6) (IC(50) 2.8 microM) exhibited strong inhibitory activity. The bis(naphtho-gamma-pyrone) derivatives did not affect the enzyme activity of inducible nitric oxide synthase (iNOS). However, these derivatives significantly reduced both the induction of iNOS protein and iNOS mRNA expression. These results suggest that the bis(naphtho-gamma-pyrone) derivatives have the pharmacologic ability to suppress NO production by activated macrophages.     

Synergistic effect of green tea catechins on cell growth and apoptosis induction in gastric carcinoma cells

(-)-Epigallocatechin gallate (EGCG), a major component of green tea catechins, is known to inhibit cell growth and to induce apoptosis in a variety of cultured cells. We examined effects of green tea catechins in cultured cells derived from human gastric carcinoma. The proliferation of four cell lines (MKN-1, MKN-45, MKN-74 and KATO-III) was inhibited with EGCG in a dose-dependent manner. The growth of MKN-45 cells was most efficiently inhibited by the treatment (IC(50): 40 muM EGCG) among the four cell lines, while KATO-III cells were most insensitive (IC(50): 80-150 muM) to the EGCG treatment. In addition, (-)-epicatechin (EC) had a major synergistic effect on the induction of apoptosis in MKN-45 cells treated with EGCG; however it had little effect on the inhibition of cell growth induced by EGCG. To study the molecular mechanisms behind the induction of apoptosis by EGCG, the activity of caspases in MKN-45 cells treated with EGCG was examined. Activity levels of caspases-3, -8 and -9 were elevated in EGCG-treated cells, suggesting that these caspases are involved in the apoptosis induced by EGCG. Furthermore, the synergistic effect of EC with EGCG on the induction of apoptosis was specifically canceled by catalase treatment, suggesting that the synergism involves the extracellular production of reactive oxygen species.     

Spatial learning of mice lacking a neuron-specific epidermal growth factor family protein, NELL2

NELL2 is a neuron-specific thrombospondin-1-like extracellular protein containing six epidermal growth factor-like domains. We previously disrupted the NELL2 gene in mice by gene targeting and showed that long-term potentiation is enhanced in vivo in the dentate gyrus of NELL2-deficient mice. To further elucidate the physiological roles of NELL2, we performed a behavioral characterization of NELL2(-/-) and their heterozygous control mice. NELL2-deficient mice exhibited learning impairment in the Morris water maze task. However, we observed no difference in passive avoidance learning between NELL2(-/-) and NELL2(+/-) mice. These observations suggest that NELL2 plays an important role in hippocampus-dependent spatial learning and that emotional learning does not depend critically on NELL2.