Screening of anti-hypoxia/reoxygenation agents by an in vitro method. Part 2: Inhibition of tyrosine kinase activation prevented hypoxia/reoxygenation-induced injury in endothelial gap junctional intercellular communication

In this study, we demonstrated that hypoxia/reoxygenation (H/R) induced an injury in gap junctional intercellular communication (GJIC) after 2 h of reoxygenation in cultured HUVEC. Free radical scavenger (DMSO) and antioxidant (SOD) did not prevent this GJIC injury at all. Protein kinase C inhibitor (calphostin C) partly blocked this injury. However, the protein tyrosine kinase (PTK) inhibitor genistein completely inhibited this GJIC injury. Compounds 1 [laxogenin-3-O-alpha-L-arabinosyl-(1-->6)- beta-D-glucopyranoside], 2 (macrostemososide A), 3 [laxogenin-3-O-beta-D-xylopyranosyl-(1-->4)-alpha- L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside], 4 (chinenoside II), 5 (beta-sitosterol), 6 (daucosterine), 7 (ginsenoside-Rd), 29 (isocumarine), 52 (icariin), 53 (icariside), and 54 (icaritin), which showed obvious influence on H/R-induced PTK activation as stated in Part 1 (except 1), were explored for their effects on GJIC. The results showed that compounds 2-7 and 52-57 partly protected H/R-induced GJIC injury. Compounds 5 and 6 (especially 5), which showed the strongest inhibitory effects on PTK activation, completely blocked H/R-provoked GJIC injury. Compound 1, which did not influence PTK activation, failed to prevent this GJIC injury. In contrast, compound 29, which significantly promoted PTK activation, enhanced this H/R-induced GJIC injury further. Western blotting of connexin 43, an important gap junctional protein for modulating GJIC in HUVEC, revealed that interference with the gap junctional protein might be the most direct mechanism for compounds 2, 5, 29, and 53 to affect H/R-injured GJIC.     

The effect of ginsenosides on adrenocorticotropin secretion in primary culture of rat pituitary cells.

The effect of ginsenoside-Rb (1), -Rc, -Rd, -Re, and Rg (1) on adrenocortiocotropin (ACTH) secretion was examined using a primary culture of pituitary cells. Ginsenoside-Rg (1), but not other ginsenosides, stimulated the secretion of ACTH. Ginsenoside-Rg (1) was found to stimulate ACTH secretion in a dose-dependent fashion; the half maximal response occurred at ca. 2.8 microM; the maximum content of ACTH was obtained at 10 microM of ginsenoside-Rg (1). The stimulation of ACTH secretion by ginsenoside-Rg (1) was observed at 15 min after the treatment. Intracellular adenosine 3', 5'-cyclic monophosphate (cyclic AMP) increased significantly at 10 minute after treatment with ginsenoside-Rg (1). Although the increased plasma corticosterone level in a rat treated with ginsenoside-Rb (1), -Rc, -Rd, and -Re is considered as the result of stimulated ACTH secretion, in this study, it is suggested that ACTH secretion is not caused by direct action of these ginsenosides to hypophysis.     

Estrogenic compounds inhibit gap junctional intercellular communication in mouse Leydig TM3 cells

Some estrogenic compounds are reported to cause testicular disorders in humans and/or experimental animals by direct action on Leydig cells. In carcinogenesis and normal development, gap junctional intercellular communication (GJIC) plays an essential role in maintaining homeostasis. In this study, we examine the effects of diethylstilbestrol (DES, a synthetic estrogen), 17beta-estradiol (E(2), a natural estrogen), and genistein (GEN, a phytoestrogen) on GJIC between mouse Leydig TM3 cells using Lucifer yellow microinjection. The three compounds tested produced GJIC inhibition in the TM3 cells after 24 h. Gradually, 10 microM DES began to inhibit GJIC for 24 h and this effect was observed until 72 h. On the other hand, both 20 microM E(2) and 25 microM GEN rapidly inhibited GJIC in 6 h and 2 h, respectively. The effects continued until 24 h, but weakened by 72 h. Furthermore, a combined effect at microM level between DES and E(2) on GJIC inhibition was observed, but not between GEN and E(2). DES and E(2) showed GJIC inhibition at low dose levels (nearly physiological estrogen levels) after 72 h, but GEN did not. DES-induced GJIC inhibition at 10 pM and 10 microM was completely counteracted by ICI 182,780 (ICl), an estrogen receptor antagonist. On the other hand, the inhibitory effects on GJIC with E(2) (10 pM and 20 microM) and GEN (25 microM) were partially blocked by ICI or calphostin C, a protein kinase C (PKC) inhibitor, and were completely blocked by the combination of ICI and calphostin C. These results demonstrate that DES inhibits GJIC between Leydig cells via the estrogen receptor (ER), and that E(2) and GEN inhibit GJIC via ER and PKC. These estrogenic compounds may have different individual non-genotoxic mechanism including PKC pathway on testicular carcinogenesis or development.     

人参皂苷Rb1对吗啡慢性作用SK-N-SH细胞增殖及[Ca~（2＋）]_i的影响

目的：探讨人参皂苷Rb1对吗啡慢性作用SK-N-SH细胞增殖及[Ca2＋]i的影响。方法：采用四甲基偶氮唑盐比色法（MTT）研究人参皂苷Rb1对吗啡慢性作用SK-N-SH细胞增殖的影响,应用荧光分光光度法观察人参皂苷Rb1对吗啡慢性作用SK-N-SH细胞内[Ca2＋]i的影响。结果：与对照组相比,吗啡慢性作用48h可显著抑制SK-N-SH细胞的增殖,细胞内游离钙离子浓度显著增高（P〈0.01）;单独加入16μmol.L-1Rb1可显著促进SK-N-SH细胞的增殖（P〈0.05）;人参皂苷Rb1（16μmol.L-1,32μmol.L-1）对细胞进行预处理能缓解吗啡对细胞的抑制作用（P〈0.05）。单独加入16μmol.L-1Rb1可使SK-N-SH细胞内[Ca2＋]i显著降低（P〈0.01）;慢性吗啡作用SK-N-SH细胞48h,能使细胞内[Ca2＋]i明显升高（P〈0.01）;慢性吗啡作用SK-N-SH后,加入（16μmol.L-1,32μmol.L-1）人参皂苷Rb1能有效地抑制吗啡引起的细胞内[Ca2＋]i升高（P〈0.01）。结论：人参皂苷Rb1可显著缓解吗啡对SK-N-SH细胞的抑制作用和吗啡引起的[Ca2＋]i升高。     

The inhibitory effects of ginsenosides on protein tyrosine kinase activated by hypoxia/reoxygenation in cultured human umbilical vein endothelial cells

27 individual ginsenosides and aglycones, together with five extracts from ginseng roots, ginseng leaves, American ginseng roots, American ginseng leaves and non-saponin fraction from roots of Panax ginseng, were tested for their effects on protein tyrosine kinase (PTK) activation induced by an in vitro hypoxia/reoxygenation (H/R) model in cultured human umbilical vein endothelial cells (HUVEC). The results indicated that ginsenoside-Rb1 (3), -Rd (7), -Ra1 (1) and -Ro (27) showed significant inhibitory effects on PTK activation induced by H/R. Dose-response experiments revealed that ginsenoside-Rb1 was the most active compound and it completely blocked PTK activation at a wide range of concentrations. Most protopanaxadiol-type ginsenosides and some protopanaxatriol-type saponins also showed significant effects on PTK activation. However, the crude extracts did not protect against H/R-induced PTK activation.     

Agonist-dependent difference in the mechanisms involved in Ca2+ sensitization of smooth muscle of porcine coronary artery

This study was undertaken to explore possible signal-transduction mechanisms involved in the Ca2+-sensitizing effects of carbachol and endothelin-1 (ET-1) by using beta-escin-skinned smooth muscle of porcine coronary artery. Pretreatment with C3 exoenzyme of Clostridium botulinum, which selectively inactivates rho p21 by adenosine diphosphate (ADP) ribosylation, resulted in a significant inhibition of ET-1-induced Ca2+ sensitization, but had no effect on carbachol-induced Ca2+ sensitization. Whereas the protein kinase C (PKC) inhibitors calphostin C and staurosporine did not affect the Ca2+-sensitizing effect of carbachol, the tyrosine kinase inhibitors genistein and tyrphostin 25 greatly but incompletely suppressed it. In contrast, the Ca2+-sensitizing effect of ET-1 was significantly inhibited by either calphostin C or genistein. Although the inhibitory effect of calphostin C on ET-1-induced Ca2+ sensitization was less than that of genistein, the effects of calphostin C and genistein were additive. The genistein-sensitive component of ET-1-induced Ca2+ sensitization appeared to include the C3-sensitive one. However, a substantial enhancement by ET-1 of the Ca2+-induced contraction was observed even in the presence of the two inhibitors. In beta-escin-skinned smooth muscle of rabbit mesenteric artery, ET-1-induced Ca2+ sensitization was marginally affected by C3 pretreatment, calphostin C, and genistein. We conclude that, although PKC activation and rho p21 protein-dependent and -independent tyrosine phosphorylation each plays an important role in an increase in myofilament Ca2+ sensitivity, the contributions of these signaling pathways to Ca2+ sensitization are different depending on receptor agonists and tissues used. Furthermore, these data suggest the existence of an as yet undefined signal-transduction mechanism involved in Ca2+ sensitization caused by receptor agonists.     

Protein kinase calpha but not p44/42 mitogen-activated protein kinase, p38, or c-Jun NH(2)-terminal kinase is required for intercellular adhesion molecule-1 expression mediated by interleukin-1beta: involvement of sequential activation of tyrosine kinase, nuclear factor-kappaB-inducing kinase, and IkappaB kinase 2

IL-1beta induced an increase in ICAM-1 expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C inhibitor (D609) attenuated IL-1beta-induced ICAM-1 expression. IL-1beta produced an increase in PKC activity and this effect was abolished by D609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited IL-1beta-induced response. TPA, a PKC activator, stimulated ICAM-1 expression as well, this effect being inhibited by tyrosine kinase inhibitors. Treatment of cells with IL-1beta resulted in stimulation of p44/42 MAPK, p38, and JNK. However, neither the mitogen activated protein kinase kinase inhibitor PD 98059 nor the p38 inhibitor SB 203580 affected IL-1beta-induced ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by IL-1beta and these effects were inhibited by tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity as well, these effects being inhibited by tyrosine kinase inhibitors. Dominant-negative PKCalpha, NIK, or IKK2, but not IKK1 mutant, inhibited IL-1beta- or TPA-induced ICAM-1 promoter activity. IKK activity was stimulated by either IL-1beta or TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. Taken together, IL-1beta activates phosphatidylcholine-specific phospholipase C and induces activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of NIK, IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression. However, activation of p44/42 MAPK, p38, and JNK is not involved.     

Effects of ginseng saponins on responses induced by various receptor stimuli

We investigated the effects of four ginseng saponins, ginsenoside-Rb1, -Rg2, -Rg3 and -Ro, on the responses induced by receptor stimulation of various stimuli. Ginsenoside-Rg2 (1-100 microM) reduced the secretions of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine and gamma-aminobutyric acid but not by angiotensin II, bradykinin, histamine and neurotensin. In guinea-pig, the ginsenoside also diminished the nicotine-induced secretion of catecholamines from the adrenal chromaffin cells, but it did not affect the muscarine- and the histamine-induced ileum contractions. On the other hand, ginsenoside-Rg3 (1-100 microM) reduced not only the acetylcholine-, the gamma-aminobutyric acid- and the neurotensin-induced secretions but also, at a higher concentration (100 microM), the angiotensin II-, the bradykinin- and the histamine-induced secretions from the bovine chromaffin cells. Furthermore, the saponin (3-100 microM) significantly inhibited the muscarine- and the histamine-induced ileum contractions of the guinea-pig. Ginsenoside-Rb1 and -Ro had no marked effect on their responses. These results strongly suggest that ginsenoside-Rg2 is a potent selective blocker of nicotinic acetylcholine and gamma-aminobutyric acid receptors (ionotropic receptors) and ginsenoside-Rg3 is not only a blocker of ionotropic receptors but also an antagonist of muscarinic or histamine receptors.     

Role of protein phosphorylation in activation of phospholipase A2 by the polychlorinated biphenyl mixture Aroclor 1242

Polychlorinated biphenyls (PCBs) activate neutrophils to induce degranulation and undergo superoxide production through a mechanism that involves stimulation of phospholipase A(2) (PLA(2)). Since the biochemical processes leading to the PCB-induced activation of this enzyme are unknown, the objective of this study was to determine whether protein phosphorylation has a role in this mechanism. Isolated rat neutrophils were labeled with [(3)H]-arachidonic acid ([(3)H]-AA), and activation of PLA(2) was determined from release of radioactivity into the medium. Exposure to the PCB mixture Aroclor 1242 induced release of [(3)H]-AA, and pretreatment with bromoenol lactone (BEL), an inhibitor of calcium-independent PLA(2), diminished release by 80%. Genistein, an inhibitor of tyrosine kinases, caused a small but significant decrease in Aroclor 1242-stimulated release of [(3)H]-AA. Daidzein, a genistein analog with no activity to inhibit tyrosine kinases, had no effect on [(3)H]-AA release. An inhibitor of p38 mitogen-activated protein kinase (MAPK), SB203580, did not affect Aroclor 1242-induced PLA(2) activity at concentrations selective for p38 MAPK; however, PD 98059, which inhibits MAPK kinase (MEK), decreased [(3)H]-AA release to about the same extent as genistein. Treatment of neutrophils with Aroclor 1242 induced phosphorylation of p44 MAPK, and this phosphorylation was unaffected by BEL but was inhibited by PD 98059. Staurosporine, a nonselective inhibitor of protein kinase C (PKC), inhibited PCB-induced release of [(3)H]-AA. Ro 32-0432, a selective inhibitor of PKC(alpha) and PKC(beta1), produced the greatest degree of inhibition (40%) among the tested protein kinase inhibitors. These results suggest that tyrosine kinases, PKC, and the MEK/MAPK pathway are involved in a fraction of Aroclor 1242-induced activation of PLA(2).     

Genistein inhibits rat aortic smooth muscle cell proliferation through the induction of p27kip1

Diet is one of the most important factors that influence the risks for cardiovascular diseases. Genistein, an isoflavone found in soy, may benefit the cardiovascular system. Here, we investigated the effect of genistein on platelet-derived growth factor (PDGF)-BB-induced proliferation of primary cultured rat aortic smooth muscle cells (RASMCs). Genistein significantly inhibited 25 ng/ml PDGF-BB-induced RASMC proliferation and [3H]-thymidine incorporation into DNA at 10, 20, and 40 microM. In accordance with these findings, genistein blocked the PDGF-BB-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Western blot analysis showed that genistein not only inhibited phosphorylation of retinoblastoma protein (pRb) and expression of cyclin E, cyclin-dependent kinase (CDK) 2, and proliferating cell nuclear antigen (PCNA) protein, but also inhibited downregulation of cyclin-dependent kinase inhibitor (CKI) p27kip1. However, genistein did not affect p21cip1, CDK4, and cyclin D1 expression or early signal transduction through PDGF beta-receptor, extracellular signal-regulated kinases 1/2 (ERK1/2), Akt, and phospholipase C (PLC) gamma1 phosphorylation. These results suggest that genistein inhibits PDGF-BB-induced RASMC proliferation via G0/G1 arrest in association with induction of p27kip1, which may contribute to the beneficial effects of genistein on the cardiovascular system.     

Uremic toxin p-cresol induces disassembly of gap junctions of cardiomyocytes

High serum levels of p-cresol have been associated with cardiovascular diseases. This study investigated the effects of p-cresol on gap junctions in neonatal cultured cardiomyocytes. p-Cresol reduced the spontaneous contraction rates of cardiomyocytes, and caused irregular cardiomyocyte beating. Junctional connexin 43 (Cx43) plaques became smaller in size and the gap junction intercellular communication (GJIC) impaired. Moreover, p-cresol increased intracellular Ca(+2) levels, and induced Ca(+2)-dependent protein kinase Cα (PKCα) activation. p-Cresol decreased P1 and P2 Cx43 levels, and increased non-phosphorylated S368-Cx43 levels. The above changes as well as Cx43 disassembly and GJIC decrease induced by p-cresol were prevented by the BAPTA-AM or PKCα inhibitor G?6976. These results suggest that PKCα mediates p-cresol-induced gap junction disassembly and GJIC dysfunction via S368-Cx43 serine dephosphorylation. This hypothesis was further confirmed in H9c2 cells by siRNA approach. SiRNA knockdown of PKCα prevented p-cresol-induced increase in nonphosphorylated Cx43. This finding supports the association of p-cresol and cardiovascular diseases.     

A New Minor Saponin from the Leaves of Panax ginseng

Seventeen compounds were isolated from the leaves of PANAX GINSENG C. A. Meyer. Among them, a new minor saponin was established as 3beta,6alpha,12beta-trihydroxy-dammar-20(22), 24-diene-6- O-alpha- L-rhamno-pyranosyl-(1-->2)-beta- D-glucopyranoside ( 2). Fourteen compounds were identified as 20( R)-protopanaxadiol ( 1), 20( R)-protopanaxatriol, ginsenoside-Rh (3), 20( R)-ginsenoside-Rh (2), 20( S)-ginsenoside-Rh (2), ginsenoside-Rh (1), -Rg (3), -Rg (2), -Rg (1), -Re, -Rd, -Rc, -Rb (2), -Rb (1); the others are still under investigation.     

20-Hydroxyeicosatetraenoic acid potentiates stretch-induced contraction of canine basilar artery via PKC alpha-mediated inhibition of KCa channel

1. The present study was undertaken to elucidate whether PKCalpha plays a role in the mechanism of the stretch-induced contraction potentiated by 20-hydroxyeicosatetraenoic acid (20-HETE). The effects of 20-HETE on the canine basilar artery were compared with those of iberiotoxin, a blocker of large conductance Ca(2+)-activated K(+) channels (K(Ca) channels), as this blocker was shown earlier to sensitize these arteries to mechanical stretch. 2. Slow stretch at rates of 0.1 to 3 mm s(-1) did not produce any contraction in normal physiological solution. 3. In the presence of 20-HETE, the slow stretch could produce contraction, which was inhibited by nicardipine, a 1,4-dihydropyridine Ca(2+) channel blocker, and gadolinium, a blocker of stretch-activated cation channels. 4. 20-HETE inhibited whole-cell K(+) current and depolarized the membrane by approximately 10 mV. These effects of 20-HETE were similar to those of iberiotoxin. 5. Calphostin C, an inhibitor of protein kinase C (PKC), inhibited the action of 20-HETE, but not that of iberiotoxin. 6. In response to 20-HETE PKCalpha isoform was translocated from the cytosol to the membrane fraction, which translocation was inhibited by calphostin C. 7. These results suggest that 20-HETE induced sensitization of the canine basilar artery to stretch was caused by PKCalpha-mediated inhibition of K(Ca) channel activity.     

Role of Ca2+-sensitive protein kinase C in phenylephrine enhancement of Ca2+ sensitivity in rat tail artery

We investigated the role of protein kinase C (PKC) isoforms on changes in sensitivity of contractile mechanisms to intracellular Ca(2+) (force /[Ca(2+)]i) by phenylephrine (0.1-100 microM) in rat tail arterial helical strips using simultaneous measurements of force and [Ca(2+)]i. Force/[Ca(2+)]Ii induced by phenylephrine was greater than that induced by 80 mM K+. Force/[Ca(2+)]i induced by phenylephrine in physiologic saline solution or low Ca(2+) solution was dependent on the agonist concentration. Removal of Ca(2+) completely abolished the phenylephrine-induced contraction. The PKC inhibitors staurosporine and calphostin C inhibited the increase in force/[Ca(2+)]i induced by phenylephrine to a much greater extent than that induced by 80 mM K+. LY379196, a specific PKCbeta inhibitor, did not inhibit the increase of calcium sensitivity due to phenylephrine. The classic PKC isoforms, alpha, betaI, and II not gamma were demonstrated in the artery by immunohistochemistry. These results suggest that in rat tail arterial smooth muscle, PKCalpha, and not beta or gamma, mediates the increase of changes in sensitivity of contractile mechanisms to intracellular Ca(2+) to high dose of alpha1 receptor stimulation (phenylephrine 100 microM) on nonphysiologic conditions.     

The potentiating effect of genistein on the relaxation induced by isoproterenol in rat aortic rings.

In rat aortic rings, the mechanism of potentiating effect of genistein, a tyrosine kinase inhibitor, on the relaxation induced by isoproterenol was examined. Pretreatment of the aortic rings by genistein, but not by daidzein, an inactive analogue of genistein, potentiated the relaxation induced by isoproterenol. Genistein also potentiated the relaxation induced by forskolin, an activator of guanylyl cyclase, and dibutyryl cyclic AMP. In addition, theophylline, an inhibitor of phosphodiesterase, potentiated the relaxation induced by isoproterenol and forskolin. Theophylline partly inhibited the potentiation of isoproterenol-induced relaxation by genistein while it completely inhibited the potentiation of forskolin-induced relaxation by genistein. Iberiotoxin, an inhibitor of Ca-activated K (KCa) channels, partly inhibited the isoproterenol-induced relaxation and the potentiating effect of genistein on the relaxation induced by isoproterenol. Quinacrine (an inhibitor of phospholipase A2), alpha-naphthoflavone (an inhibitor of cytochrome P-450 enzymes), and 8-methoxypsoralen (an inhibitor of cytochrome P-450 enzymes), partly inhibited the potentiating effect of genistein on the isoproterenol-induced relaxation, but metyrapone (an inhibitor of cytochrome P-450 enzymes), indomethacin (an inhibitor of cyclooxygenase), and AA861 (an inhibitor of 5-lipoxygenase) did not. These results suggest that the potentiation of isoproterenol-induced relaxation by genistein may be related to the activities of phosphodiesterase, KCa channels, and cytochrome P-450 enzymes.     

Contribution of Ca(2+) -dependent protein kinase C in the spinal cord to the development of mechanical allodynia in diabetic mice

In this paper, we directly demonstrate, for the first time, the activation of Ca(2+)-dependent protein kinase C (PKC) in the spinal cord of diabetic mice. In streptozotocin (STZ)-treated (200 mg/kg, i.v.) diabetic mice, hypersensitivity (allodynia) to mechanical stimulation appeared 7 d after STZ injection. This mechanical allodynia was inhibited by intrathecal injection of the PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, but not the protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89). The activity of membrane-associated Ca(2+)-dependent PKC in the spinal cords of STZ-induced diabetic mice was significantly higher than that observed in non-diabetic mice. These results suggest that activation of Ca(2+)-dependent PKC in the spinal cord, contributes to the mechanical allodynia in the pain associated with diabetic neuropathy.     

Effects of ginseng total saponins on pacemaker currents of interstitial cells of Cajal from the small intestine of mice

Although ginsenosides have a variety of physiologic or pharmacologic functions in various regions, there are only a few reports on the effects of ginsenosides on gastrointestinal (GI) motility. We studied the modulation of pacemaker activities by ginseng total saponins in the interstitial cells of Cajal (ICC) using the whole cell patch-clamp technique. Externally applied ginseng total saponins (GTS) produced membrane depolarization in the current-clamp mode and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid or niflumic acid abolished the generation of pacemaker currents, but only treatment with flufenamic acid inhibited the GTS-induced tonic inward currents. The tonic inward currents induced by GTS were not inhibited by the intracellular application of guanosine 5'-[beta-thio]diphosphate trilithium salt. Pretreatment with a Ca(2+)-free solution, with U-73122, an active phospholipase C inhibitor, and with thapsigargin, a Ca(2')-ATPase inhibitor of the endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the GTS-induced action. However, treatment with chelerythrine and calphostin C, protein kinase C inhibitors, did not block the GTS-induced effects on the pacemaker currents. These results suggest that ginsenosides modulate the pacemaker activities of the ICC, and the ICC can be targets for ginsenosides, and their interaction can affect intestinal motility.     

Mechanism of endothelial nitric oxide-dependent vasorelaxation induced by wine polyphenols in rat thoracic aorta

The mechanisms by which red wine polyphenolic compounds (RWPCs) induced endothelium-dependent relaxation were investigated in rat thoracic aorta rings with endothelium. RWPCs produced relaxation that was prevented by the nitric oxide (NO) synthase inhibitor, N(omega)-nitro-L-arginine-methyl-ester. This relaxation was abolished in the absence of extracellular calcium in the medium or in the presence of the Ca2+ entry blocker, La3+, but it was not affected by the nonselective K+ channels blocker, tetrabutylammonium. N-Ethyl-maleimide (NEM), a sulfhydryl alkylating agent, abolished vasorelaxation produced by RWPCs and acetylcholine but not that produced either by the sarcoendoplasmic reticulum Ca2+-adenosine triphosphatase (ATPase) pump inhibitor, cyclopyazonic acid (CPA) or the calcium ionophore, ionomycin. Neither pertussis toxin (PTX) nor cholera toxin (CTX) inhibited the vasorelaxant effect of RWPC. The effect of RWPC was not affected by the phospholipase C (PLC) blocker, L-alpha-glycerophospho-D-myo-inositol 4-monophosphate (Gro-pip), and the phospholipase A2 pathway blockers, quinacrine and ONO-RS-082. Finally, the protein kinase C (PKC) inhibitor, GF 109203X, and tyrosine kinase inhibitors, tyrphostin A-23 and genistein, did not impair the response to RWPCs. These results suggest that RWPCs produce endothelium-NO-derived vasorelaxation through an extracellular Ca2+-dependent mechanism via an NEM-sensitive pathway. They also show that PTX- or CTX-sensitive G proteins, activation of PLC or PLA2 pathways, PKC, or tyrosine kinase may not be involved.