Wing-Hung Ko

Vasorelaxant and antiproliferative effects of berberine.

The present study was intended to examine the relaxant effects of berberine in rat isolated mesenteric arteries. Berberine produced a rightward shift of the concentration-response curve to phenylephrine and significantly reduced the maximal contractile response to phenylephrine. Berberine (10(-7)-3x10(-5) M) also relaxed the phenylephrine- and 9,11-dideoxy-11alpha, 9alpha-epoxy-methanoprostaglandin F(2alpha)-precontracted arteries with respective IC(50) values of 1.48+/-0.16x10(-6) and 2.23+/-0. 22x10(-6) M. Removal of a functional endothelium significantly attenuated the berberine-induced relaxation (IC(50): 4.73+/-0. 32x10(-6) M) without affecting the maximum relaxant response. Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME) or methylene blue reduced the relaxant effect of berberine, and L-arginine (10(-3) M) partially antagonized the effect of L-NAME. In contrast, pretreatment with 10(-6) M glibenclamide or 10(-5) M indomethacin had no effect. Berberine (10(-5) M) reduced over by 50% the transient contraction induced by caffeine or phenylephrine in endothelium-denuded rings bathed in Ca(2+)-free Krebs solution. Pretreatment with putative K(+) channel blockers, such as tetrapentylammonium ions (1-3x10(-6) M), 4-aminopyridine (10(-3) M), or Ba(2+) (3x10(-4) M), significantly attenuated the berberine-induced relaxation in endothelium-denuded arteries. In contrast, tetraethylammonium ions (3x10(-3) M), charybdotoxin (10(-7) M) or glibenclamide (10(-6) M) were without effect. Berberine reduced the high-K(+)-induced sustained contraction and the relaxant response to berberine was greater in rings with endothelium (IC(50): 4.41+/-0.47x10(-6) M) than in those without endothelium (IC(50): 8.73+/-0.74x10(-6) M). However, berberine (10(-6)-10(-4) M) did not affect the high-K(+)-induced increase of intracellular [Ca(2+)] in cultured aortic smooth muscle cells. Berberine did not affect active phorbol ester-induced contraction in Ca(2+)-free Krebs solution. In addition, berberine inhibited proliferation of cultured rat aortic smooth muscle cells with an IC(50) of 2.3+/-0.43x10(-5) M. These findings suggest that berberine could act at both endothelium and the underlying vascular smooth muscle to induce relaxation. Nitric oxide from endothelium may account primarily for the berberine-induced endothelium-dependent relaxation, while activation of tetrapentylammonium-, 4-aminopyridine- and Ba(2+)-sensitive K(+) channels, inhibition of intracellular Ca(2+) release from caffeine-sensitive pools, or a direct relaxant effect, is likely responsible for the berberine-induced endothelium-independent relaxation. Mechanisms related to either Ca(2+) influx or protein kinase C activation may not be involved. Both vasorelaxant and antiproliferative effects may contribute to a long-term benefit of berberine in the vascular system.

Halogenated silicon(IV) phthalocyanines with axial poly(ethylene glycol) chains. Synthesis, spectroscopic properties, complexation with bovine serum albumin and in vitro photodynamic activities

A new series of unsubstituted and halogenated silicon(IV) phthalocyanines with two axial poly(ethylene glycol) (PEG) chains having an average molecular weight of 550 or 750 (PEG550 or PEG750) have been synthesised by treating the corresponding silicon phthalocyanine dichloride with PEG methyl ether in the presence of NaH. The compounds have been unambiguously characterised with 1H NMR and MALDI-TOF mass spectrometry. With two bulky polymeric substituents, the compounds are essentially non-aggregated in common organic solvents. The longer PEG750 chain enhances the hydrophilicity of the phthalocyanine ring and is more effective to prevent aggregation and fluorescence quenching by Cu(OAc)2. Substitution with heavier halogen atoms on the periphery of the ring leads to a reduction in fluorescence emission and an increase in singlet oxygen quantum yield, as a result of heavy atom effect. The compounds Si(PcX8)(PEG750)2 [X = H (4b), Cl (4c), Br (4d)] are photocytotoxic towards HepG2 human hepatocarcinoma cells and J774 mouse mammary tumour cells. Although halogenation results in an increase in singlet oxygen quantum yield, the general photocytotoxicity follows the order 4b > 4d > 4c. This can be attributed to the opposite effect of aggregation, which follows the order 4a < 4b < 4c in the growth medium. The interactions of 4b–d with bovine serum albumin (BSA) have also been investigated by a fluorescence quenching method and a non-covalent conjugate of 4b and BSA has been prepared. Conjugation with BSA leads to a higher photocytotoxicity against J774 cells, which have a BSA-loving macrophage origin.

Effects of Cordyceps sinensis, Cordyceps militaris and their isolated compounds on ion transport in Calu-3 human airway epithelial cells

AIM OF THE STUDY The traditional Chinese medicine Cordyceps sinensis (CS) (Clavicipitaceae) improves pulmonary function and is used to treat respiratory disease. Here, we compare the efficacy and mechanisms of action of Cordyceps sinensis and Cordyceps militaris (CM) (Clavicipitaceae) in Calu-3 human airway epithelial monolayer model. MATERIAL AND METHODS The extracts of Cordyceps sinensis and Cordyceps militaris, as well as their isolated compounds, cordycepin and adenosine, stimulated ion transport in a dose-dependent manner in Calu-3 monolayers. In subsequent experiments, transport inhibitor bumetanide and carbonic anhydrase inhibitor acetazolamide were added after Cordyceps sinensis and Cordyceps militaris extracts to determine their effects on Cl- and HCO3- movement. RESULTS The results suggested that Cordyceps sinensis and Cordyceps militaris extracts may affect the anion movement from the basolateral to apical compartments in the airway epithelia. CONCLUSIONS Basolateral Na+-K+-2Cl- cotransporter and apical cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channel are involved in the process. The results provide the first evidence for the pharmacological mechanism of Cordyceps sinensis and Cordyceps militaris on respiratory tract.

Cellular Mechanisms Underlying the Laxative Effect of Flavonol Naringenin on Rat Constipation Model

Background & Aims Symptoms of constipation are extremely common, especially in the elderly. The present study aim to identify an efficacious treatment strategy for constipation by evaluating the secretion-promoting and laxative effect of a herbal compound, naringenin, on intestinal epithelial anion secretion and a rat constipation model, respectively. Methods/Principal Findings In isolated rat colonic crypts, mucosal addition of naringenin (100 µM) elicited a concentration-dependent and sustained increase in the short-circuit current (ISC), which could be inhibited in Cl− free solution or by bumetanide and DPC (diphenylamine-2-carboxylic acid), but not by DIDS (4, 4′- diisothiocyanatostilbene-2, 2′-disulfonic acid). Naringenin could increase intracellular cAMP content and PKA activity, consisted with that MDL-12330A (N-(Cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride) pretreatment reduced the naringenin-induced ISC. In addition, significant inhibition of the naringenin-induced ISC by quinidine indicated that basolateral K+ channels were involved in maintaining this cAMP-dependent Cl− secretion. Naringenin-evoked whole cell current which exhibited a linear I–V relationship and time-and voltage- independent characteristics was inhibited by DPC, indicating that the cAMP activated Cl− conductance most likely CFTR (cystic fibrosis transmembrane conductance regulator) was involved. In rat constipation model, administration of naringenin restored the level of fecal output, water content and mucus secretion compared to loperamide-administrated group. Conclusions Taken together, our data suggest that naringenin could stimulate Cl− secretion in colonic epithelium via a signaling pathway involving cAMP and PKA, hence provide an osmotic force for subsequent colonic fluid secretion by which the laxative effect observed in the rat constipation model. Naringenin appears to be a novel alternative treatment strategy for constipation.

Polarized Secretion of Interleukin (IL)-6 and IL-8 by Human Airway Epithelia 16HBE14o- Cells in Response to Cationic Polypeptide Challenge

Background The airway epithelium participates in asthmatic inflammation in many ways. Target cells of the epithelium can respond to a variety of inflammatory mediators and cytokines. Damage to the surface epithelium occurs following the secretion of eosinophil-derived, highly toxic cationic proteins. Moreover, the surface epithelium itself is responsible for the synthesis and release of cytokines that cause the selective recruitment, retention, and accumulation of various inflammatory cells. To mimic the damage seen during asthmatic inflammation, the bronchial epithelium can be challenged with highly charged cationic polypeptides such as poly-l-arginine. Methodology/Principal Findings In this study, human bronchial epithelial cells, 16HBE14o- cells, were “chemically injured” by exposing them to poly-l-arginine as a surrogate of the eosinophil cationic protein. Cytokine antibody array data showed that seven inflammatory mediators were elevated out of the 40 tested, including marked elevation in interleukin (IL)-6 and IL-8 secretion. IL-6 and IL-8 mRNA expression levels were elevated as measured with real-time PCR. Cell culture supernatants from apical and basolateral compartments were collected, and the IL-6 and IL-8 production was quantified with ELISA. IL-6 and IL-8 secretion by 16HBE14o- epithelia into the apical compartment was significantly higher than that from the basolateral compartment. Using specific inhibitors, the production of IL-6 and IL-8 was found to be dependent on p38 MAPK, ERK1/2 MAPK, and NF-κB pathways. Conclusions/Significance The results clearly demonstrate that damage to the bronchial epithelia by poly-l-arginine stimulates polarized IL-6 and IL-8 secretion. This apically directed secretion of cytokines may play an important role in orchestrating epithelial cell responses to inflammation.

Depletion of intracellular Ca2+ stores enhances flow-induced vascular dilatation in rat small mesenteric artery.

The effect of depleting intracellular Ca2+ stores on flow‐induced vascular dilatation and the mechanism responsible for the vasodilatation were examined in rat isolated small mesenteric arteries. The arteries were pressurized to 50 mmHg and preconstricted with phenylephrine. Intraluminal flow reversed the effect of phenylephrine, resulting in vasodilatation. Flow dilatation consisted of an initial transient peak followed by a sustained plateau phase. The magnitude of dilatation was markedly reduced by removing Ca2+ from the intraluminal flow medium. Depletion of intracellular Ca2+ stores with either cyclopiazonic acid (CPA, 2 μM) or 1,4‐dihydroxy‐2,5‐di‐tert‐butylbenzene (BHQ, 10 μM) significantly augmented the magnitude of flow dilatation. Flow‐induced endothelial cell Ca2+ influx was also markedly enhanced in arteries pretreated with CPA or BHQ. Flow‐induced dilatation was insensitive to Nw‐nitro‐L‐arginine methyl ester (100 μM) plus indomethacin (3 μM) or to oxyhemoglobin (3 μM), but was markedly reduced by 30 mM extracellular K+ or 2 mM tetrabutylammonium (TBA), suggesting an involvement of EDHF. Catalase at 1200 U ml−1 abolished the flow‐induced dilatation, while the application of exogenous H2O2 (90–220 μM) induced relaxation in phenylephrine‐preconstricted arteries. Relaxation to exogenous H2O2 was blocked in the presence of 30 mM extracellular K+, and H2O2 (90 μM) hyperpolarized the smooth muscle cells, indicating that H2O2 can act as an EDHF. In conclusion, flow‐induced dilatation in rat mesenteric arteries can be markedly enhanced by prior depletion of intracellular Ca2+ stores. Furthermore, these data are consistent with a role for H2O2 as the vasodilator involved.

Regulation of Cl− Secretion by Extracellular ATP in Cultured Mouse Endometrial Epithelium

Abstract. The present study explored regulation of electrogenic ion transport across cultured mouse endometrial epithelium by extracellular ATP using the short-circuit current (ISC) and the patch-clamp techniques. The cultured endometrial monolayers responded to apical application of ATP with an increase in ISC in a concentration-dependent manner (EC50 at 3 μm). Replacement of Cl− in the bathing solution or treatment of the cells with Cl− channel blockers, DIDS and DPC, markedly reduced the ISC, indicating that a substantial portion of the ATP-activated ISC was Cl−-dependent. Amiloride at a concentration (10 μm) known to block Na+ channels was found to have no effect on the ATP-activated ISC excluding the involvement of Na+ absorption. Adenosine was found to have little effect on the ISC excluding the involvement of P1 receptors. The effect of UTP, a potent P2U receptor agonist on the ISC was similar to that of ATP while potent P2X agonist, α-β-Methylene adenosine 5′-triphosphate (α-β-M-ATP) and P2Y agonist, 2-methylthio-adenosine triphosphate (2-M-ATP), were found to be ineffective. The effect of ATP on ISC was mimicked by the Ca2+ ionophore, ionomycin, indicating a role of intracellular Ca2+ in mediating the ATP response. Confocal microscopic study also demonstrated a rise in intracellular Ca2+ upon stimulation by extracellular ATP. In voltage-clamped endometrial epithelial cells, ATP elicited a whole-cell Cl− current which exhibited outward rectification and delayed activation and inactivation at depolarizing and hyperpolarizing voltages, respectively. The results of the present study demonstrate the presence of a regulatory mechanism involving extracellular ATP and P2U purinoceptors for endometrial Cl− secretion.

Expression, Immunolocalization, and Functional Activity of Na+/H+ Exchanger Isoforms in Mouse Endometrial Epithelium

Abstract The luminal fluid microenvironment of the uterus is important for sperm capacitation and embryo development. In an attempt to understand the possible role of Na+/H+ exchangers (NHEs) in uterine function, the mRNAs of different NHE isoforms as well as their subcellular localization (apical versus basolateral) and functional activity were investigated in mouse endometrial epithelial cells using reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and intracellular pH (pHi) measurement techniques. The presence of NHE1, NHE2, and NHE4, but not NHE3 mRNAs were revealed by RT-PCR. Immunostaining showed that NHE1, NHE2, and NHE4 were present in both apical and basolateral membranes. The pHi recovery from intracellular acidification was Na+-dependent; however, the rate of pHi recovery depending on basolateral Na+ was 12.4 times faster than that depending on apical Na+. The Na+-dependent rate of pHi recovery was also inhibited by amiloride, indicating H+ extrusion through NHEs; however, the amiloride sensitivity of the apical membrane was less than that of the basolateral membrane, suggesting the involvement of different types of NHEs in the two membranes. The results indicate that the basolaterally located NHE1, NHE2, and NHE4, in addition to participating in the homeostatic control of intracellular pH, may play a role in H+ extrusion in order to achieve transepithelial HCO3− secretion. The apically located NHEs may be involved in mediating Na+ absorption as alternatives of or complementary to epithelial Na+ channels.

Regulated anion secretion in cultured epithelia from Sertoli cells of immature rats

1 Cultured epithelia of Sertoli cells from prepubertal rats were grown on Matrigel‐coated millipore filters for short‐circuit current (Isc) measurements. Under basal conditions, these epithelia exhibited a ‘zero’ transepithelial potential difference, a ‘zero’ short‐circuit current and a transepithelial resistance of 60 Ω cm2. 2 Forskolin (100 μm) and 8‐(4‐chlorophenylthio)‐cAMP (cpt‐cAMP) (100 μm) added to the apical side stimulated the Isc (forskolin, peak ΔIsc= 1.32 ± 0.16 μA cm−1; cpt‐cAMP, peak ΔIsc= 0.88 ± 0.16 μA cm−2). 3 ATP (100 μm) added apically elicited a Isc response (peak ΔIsc= 6.45 ± 0.28 μA cm−2) which was similar in magnitude to that of 1 μm thapsigargin (peak ΔIsc= 6.09 ± 0.44 μA cm−2). The potency of the responses to other nucleotides: UTP ≥ ATP > ADP >> AMP = adenosine indicates the involvement of a mixture of P2Y receptors. 4 Removal of extracellular Cl− and HCO3− reduced the Isc response to ATP by 70 % and 40 %, respectively. Removal of K+ had no effect, whereas removal of Na+ attenuated the Isc response. 5 The response to ATP was insensitive to agents known to block anion secretion (except apical diphenylamine‐2‐carboxylate (DPC) and DIDS). The resistance to perturbation by pharmacological agents may be a unique property of the seminiferous epithelium. 6 Whole‐cell current recordings in cultured rat Sertoli cells demonstrated a DIDS‐sensitive outwardly rectifying Cl− conductance with activating and inactivating characteristics at depolarizing and hyperpolarizing voltages, respectively. 7 The stimulation of electrogenic ion transport by ATP may be part of a complex mechanism regulating fluid secretion by the testis. Cultured Sertoli cell epithelia are shown to provide a useful model to investigate transepithelial transport in the seminiferous epithelium.

BAM-SiPc, a novel agent for photodynamic therapy, induces apoptosis in human hepatocarcinoma HepG2 cells by a direct mitochondrial action.

Photodynamic therapy is recently developed as an effective treatment for malignant disease. The therapeutic effect depends on the properties of the photosensitizers. Among the novel photosensitizers we have synthesized, the unsymmetrical bisamino phthalocyanine, SiPc[C3H5(NMe2)2O](OMe) [abbreviated as BAM-SiPc] is particularly active in the HepG2 cell culture model. Fluorescence microscopy has also indicated that it targets the mitochondria. In the present investigation, the biochemical mechanisms of BAM-SiPc leading to cell death were investigated. Photodynamic treatment with BAM-SiPc resulted in the generation of reactive oxygen species and a collapse of mitochondrial membrane potential. The pro-apoptotic Bax protein was translocated from the cytosol to mitochondria; while the level of the mitochondrial anti-apoptotic Bcl-2 protein decreased after photodynamic treatment. Cytochrome c, but not apoptosis-inducing factor, was released from the mitochondria into the cytosol, subsequently resulting in the cleavage of poly(ADP-ribose) polymerase. These events were at least partially responsible for the observed BAM-SiPc induced apoptosis, which was clearly demonstrated by (a) the loss of membrane asymmetry, (b) DNA ladder formation, and (c) the presence of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells.