Sensitive Detection of Ciguatoxins Using a Neuroblastoma Cell-Based Assay with Voltage-Gated Potassium Channel Inhibitors.

Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 µg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (NaV channels) and blocking of voltage-gated potassium channels (KV channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using KV channel inhibitors as sensitizing reagents for N2a cells. The addition of the KV channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of KV channels on the toxicity of CTXs in a cell-based assay.

A density-tunable liquid-phase microextraction system based on deep eutectic solvents for the determination of polycyclic aromatic hydrocarbons in tea, medicinal herbs and liquid foods.

A novel density-tunable liquid-phase microextraction (DT-LPME) system was developed with high-density deep eutectic solvents (DESs) as extractant and low-density organic solvents as emulsifier and density regulator. DES-rich phase was induced to form in the bottom or in the top by adjusting the emulsifier amount. This system was used to directly extract polycyclic aromatic hydrocarbons (PAHs) from liquid and solid foods, and the obtained DES-rich phase was easy to be collected for quantification. The method (LPME with HPLC-fluorescence detector) has linearity (R2 > 0.9974), detection limits of 0.6-4.2 ng L-1 for liquid foods and 0.05-0.35 ng g-1 for solid foods, recoveries of 86.2-114.9%, and intra-day/inter-day RSDs below 6.6%. The method was applied to detect PAHs in real samples, and the PAHs residue was found in honey and five solid foods. The DT-LPME method is simple, fast, green and suitable for direct extraction of analytes from both liquid and solid samples.

Major flavonoids from Psiadia punctulata produce vasodilation via activation of endothelial dependent NO signaling.

Vasodilators are important pharmacologic agents for managing and/or treating hypertension. Medicinal plants are considered as valuable source of bioactive compounds. We used a bioguided approach to isolate, identify, and investigate the possible vasodilation activities and mechanism(s) of the prepared methanol extract from aerial parts of Psiadia punctulata (MAPP), its bioactive fraction and active compounds. Vascular effects of MAPP were studied using isolated artery technique in the presence or absence of specific candidate pathways inhibitors, and found to produce a significant vasodilation of phenylephrine preconstricted rat aortae. The bioactive chloroform fraction yielded five methoxylated flavonoids: umuhengerin (1), gardenin A (2), gardenin B (3), luteolin-3',4' -dimethyl ether (4), and 5,3'-dihydroxy-6,7,4',5'-tetramethoxyflavone (5). Metabolites 1, 4, and 5 produced a significant vasodilation. Removal of the endothelium significantly inhibited MAPP vasodilation. Nitric oxide synthase inhibition and not prostacycline inhibition or K+ channel blocking, was found to cause the observed vasodilation inhibition. Both guanylate cyclase and adenylate cyclase inhibitions markedly inhibited MAPP vasodilation. In conclusion MAPP possesses vasodilation activities that is mediated through endothelial nitric oxide pathway, calcium dependent endothelial nitric oxide synthase activation, and interference with the depolarization process through calcium channel blocking activity.

Vasorelaxant Effect of Novel Nitric Oxide-Hydrogen Sulfide Donor Chalcone in Isolated Rat Aorta: Involvement of cGMP Mediated sGC and Potassium Channel Activation.

BACKGROUND AND OBJECTIVE: Recently, nitric oxide (NO) and hydrogen sulfide (H2S) donating moieties were extensively studied for their role in the vasculature as they are responsible for many cellular and pathophysiological functioning. The objective of the present study is to evaluate novel NO and H2S donating chalcone moieties on isolated rat aorta for vasorelaxation, and to investigate the probable mechanism of action. METHODS: To extend our knowledge of vasorelaxation by NO and H2S donor drugs, here we investigated the vasorelaxing activity of novel NO and H2S donating chalcone moieties on isolated rat aorta. The mechanism of vasorelaxation by these molecules was investigated by performing in vitro cGMP mediated sGC activation assay and using Tetraethylammonium chloride (TEA) as a potassium channel blocker and Methylene blue as NO blocker. RESULTS: Both NO and H2S donating chalcone moieties were found to be potent vasorelaxant. The compound G4 and G5 produce the highest vasorelaxation with 3.716 and 3.789 M of pEC50, respectively. After the addition of TEA, G4 and G5 showed 2.772 and 2.796 M of pEC50, respectively. The compounds Ca1, Ca2, and D7 produced significant activation and release of cGMP mediated sGC which was 1.677, 1.769 and 1.768 M of pEC50, respectively. CONCLUSION: The vasorelaxation by NO-donating chalcones was blocked by Methylene blue but it did not show any effect on H2S donating chalcones. The vasorelaxing potency of NO-donating molecules was observed to be less affected by the addition of TEA but H2S donors showed a decrease in both efficacy and potency. The cGMP release was more in the case of NO-donating molecules. The tested compounds were found potent for relaxing vasculature of rat aorta.

Voltage-gated potassium channels are critical for infrared inhibition of action potentials: an experimental study.

Thermal block of unmyelinated axons may serve as a modality for control, suggesting a means for providing therapies for pain. Computational modeling predicted that potassium channels are necessary for mediating thermal block of propagating compound action potentials (CAPs) with infrared (IR) light. Our study tests that hypothesis. Results suggest that potassium channel blockers disrupt the ability of IR to block propagating CAPs in Aplysia californica nerves, whereas sodium channel blockers appear to have no significant effect. These observations validate the modeling results and suggest potential applications of thermal block to many other unmyelinated axons.

Vasoconstriction triggered by hydrogen sulfide: Evidence for Na+,K+,2Cl-cotransport and L-type Ca2+ channel-mediated pathway.

OBJECTIVES: This study examined the dose-dependent actions of hydrogen sulfide donor sodium hydrosulphide (NaHS) on isometric contractions and ion transport in rat aorta smooth muscle cells (SMC). METHODS: Isometric contraction was measured in ring aortas segments from male Wistar rats. Activity of Na+/K+-pump and Na+,K+,2Cl-cotransport was measured in cultured endothelial and smooth muscle cells from the rat aorta as ouabain-sensitive and ouabain-resistant, bumetanide-sensitive components of the 86Rb influx, respectively. RESULTS: NaHS exhibited the bimodal action on contractions triggered by modest depolarization ([K+]o=30 mM). At 10-4 M, NaHS augmented contractions of intact and endothelium-denuded strips by ~ 15% and 25%, respectively, whereas at concentration of 10-3 M it decreased contractile responses by more than two-fold. Contractions evoked by 10-4 M NaHS were completely abolished by bumetanide, a potent inhibitor of Na+,K+,2Cl-cotransport, whereas the inhibition seen at 10-3 M NaHS was suppressed in the presence of K+ channel blocker TEA. In cultured SMC, 5×10-5 M NaHS increased Na+,K+,2Cl- - cotransport without any effect on the activity of this carrier in endothelial cells. In depolarized SMC, 45Ca influx was enhanced in the presence of 10-4 M NaHS and suppressed under elevation of [NaHS] up to 10-3 M. 45Ca influx triggered by 10-4 M NaHS was abolished by bumetanide and L-type Ca2+ channel blocker nicardipine. CONCLUSIONS: Our results strongly suggest that contractions of rat aortic rings triggered by low doses of NaHS are mediated by activation of Na+,K+,2Cl-cotransport and Ca2+ influx via L-type channels.

Toxicology of potassium channel-directed compounds in mosquitoes.

Potential targets for new vector control insecticides are nerve and muscle potassium channels. In this study, the activities of known potassium channel blockers (4-aminopyridine, quinidine, and tetraethylammonium) and the insecticide propoxur were compared to three experimental catechols and several other compounds against Anopheles gambiae and Aedes aegypti mosquitoes. Experimental catechol 1 was the most toxic experimental compound in all of the mortality assays conducted, but was at least 100-fold and 39-fold less toxic than propoxur against Ae. aegypti and An. gambiae, respectively. Injection treatment and synergist (piperonyl butoxide) bioassays found that catechol toxicity was not unduly impacted by cuticular transport or oxidative metabolism. Electrophysiological studies showed a decrease in amplitude of evoked muscle contractions, along with an increase in twitch duration at concentrations that increased basal muscle tension (mM). High concentration effects on basal muscle tension were matched by complete depolarization of the muscle membrane potential. Effects on muscle physiology and blockage of Kv2.1 potassium channels in patch clamp experiments were generally consistent with in vivo toxicity, except for 4-aminopyridine, which suggest the involvement of other potassium channel subtypes. Extensive melanization of Anopheles larvae, but not Aedes larvae, occurred from exposure to catechol compounds. Interaction with the phenol oxidase system within insects may be the cause of this melanization, but any contribution to toxicity requires further investigation.

Tertiary amine mediated targeted therapy against metastatic lung cancer.

In this work, two tertiary amine-derived 4'-demethylepipodophyllotoxin (DMEP) conjugates (DC and DP) have been designed and synthesized using N,N,N'-trimethyl-N'-(4-carboxyl benzyl)-1,3-propanediamine (CPDM) and 4-(4-methylpiperazinomethyl)benzoic acid (PBA) as the targeting ligands. Both DC and DP exhibited strong in vitro cytotoxicity against small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines. Cellular uptake efficiencies of DC and DP in human alveolar type II epithelial cells were significantly enhanced compared to DMEP and etoposide (VP-16), which were demonstrated to be concentration-, time- and energy-dependent. The active transport process of DC and DP might be mediated by organic cation transporters (OCTs). After systemic administration in mice, both DC and DP selectively accumulated in the lung, displaying the highest Cmax and AUC0-t values of all tested tissues. Compared with DMEP and VP-16, DC and DP remarkably reduced the lung weight and the number of lung metastases of B16 melanoma in mice, and further prolonged the survival of tumor-bearing mice. Also, DC and DP exhibited comparable levels of cell cycle arrest and cell apoptosis. Furthermore, DC and DP demonstrated minimum toxicity towards vital organs and reduced gastrointestinal injury compared to DMEP and VP-16. Taken together, tertiary amine-derived moieties such as CPDM and PBA represent an efficient yet safe strategy to achieve lung-targeted drug delivery.

Nitric oxide and Kir6.1 potassium channel mediate isoquercitrin-induced endothelium-dependent and independent vasodilation in the mesenteric arterial bed of rats.

The vascular effect of flavonoid isoquercitrin was investigated in the perfused mesenteric vascular bed of rats. In preparations with functional endothelium isoquercitrin (100, 300 and 1000nmol) dose-dependently reduced the perfusion pressure by 13±2.2, 33±3.9, and 58±3.7mm Hg, respectively. Endothelium removal or inhibition of the nitric oxide synthase enzymes by l-NAME did not change the effects of 100 and 300 nmol isoquercitrin, but reduced by 30-40% the vasodilation induced by 1000 nmol isoquercitrin. Perfusion with nutritive solution containing 40mM KCl abolished the vasodilatory effect of all isoquercitrin doses. Treatment with glibenclamide, a Kir6.1 (ATP-sensitive) potassium channel blocker, inhibited vasodilation induced by 100 and 300 nmol isoquercitrin, but only partially reduced the effect of 1000 nmol isoquercitrin. The non-selective KCa (calcium-activated) potassium channel blocker tetraethylammonium, but not the selective KCa1.1 channel blocker iberiotoxin, reduced by around 60% vasodilation induced by all isoquercitrin doses. In addition, association of tetraethylammonium and glibenclamide, or l-NAME and glibenclamide, fully inhibited isoquercitrin-induced vasodilation. Our study shows that isoquercitrin induces vasodilation in resistance arteries, an effect mediated by K(+) channel opening and endothelial nitric oxide production.

Inhibition of voltage-gated potassium channels mediates uncarboxylated osteocalcin-regulated insulin secretion in rat pancreatic ß cells.

Insulin secretion from pancreatic ß cells is important to maintain glucose homeostasis and is regulated by electrical activities. Uncarboxylated osteocalcin, a bone-derived protein, has been reported to regulate glucose metabolism by increasing insulin secretion, stimulating ß cell proliferation and improving insulin sensitivity. But the underlying mechanisms of uncarboxylated osteocalcin-modulated insulin secretion remain unclear. In the present study, we investigated the relationship of uncarboxylated osteocalcin-regulated insulin secretion and voltage-gated potassium (KV) channels, voltage-gated calcium channels in rat ß cells. Insulin secretion was measured by radioimmunoassay. Channel currents and membrane action potentials were recorded using the conventional whole-cell patch-clamp technique. Calcium imaging system was used to analyze intracellular Ca(2+) concentration ([Ca(2+)]i). The data show that under 16.7mmol/l glucose conditions uncarboxylated osteocalcin alone increased insulin secretion and [Ca(2+)]i, but with no such effects on insulin secretion and [Ca(2+)]i in the presence of a KV channel blocker, tetraethylammonium chloride. In the patch-clamp experiments, uncarboxylated osteocalcin lengthened action potential duration and significantly inhibited KV currents, but had no influence on the characteristics of voltage-gated calcium channels. These results indicate that KV channels are involved in uncarboxylated osteocalcin-regulated insulin secretion in rat pancreatic ß cells. By inhibiting KV channels, uncarboxylated osteocalcin prolongs action potential duration, increases intracellular Ca(2+) concentration and finally promotes insulin secretion. This finding provides new insight into the mechanisms of osteocalcin-modulated insulin secretion.

Inhibitory efficacy of bufadienolides on Na+,K+-pump activity versus cell proliferation.

Bufadienolides are cytotoxic drugs that may form the basis for anticancer agents. Due to structural and functional similarity to cardiotonic glycosides, application is restricted. We, therefore, investigated correlation of their putative anticancer effects with inhibition of Na+,K+pumps. The natural bufalin and three derivatives were tested. The anticancer effects of the drugs were checked by observing their inhibitory effects on proliferation of rat liver cancer cells using MTT assay. Inhibition of Na+,K+-pump was determined by measuring pump-mediated current of rat α1/ß1 and α2/ß1 Na+,K+pumps expressed in Xenopus oocytes. All tested bufadienolides inhibited cell proliferation and Na+,K+pump activity. An activity coefficient A=100xIC50Na,K pump/IC50proliferation was used to describe drug effectivity as anticancer drug. Natural bufalin exhibited lowest effectivity on cell proliferation, and also the A value for rat α1 isoform was the lowest (0.08), the α2 isoform was much less sensitive (A=1.00). The highest A values were obtained for the BF238 derivative with A=0.88 and 2.64 for the α1 and α2 isoforms, respectively. Therefore, we suggest that search for bufalin derivatives with high anticancer effect and low affinity for both Na+,K+pump isoforms may be a promising strategy for development of anticancer drugs.

Flavonoid galetin 3,6-dimethyl ether attenuates guinea pig ileum contraction through K(+) channel activation and decrease in cytosolic calcium concentration.

Flavonoid galetin 3,6-dimethyl ether (FGAL) has been isolated from the aerial parts of Piptadenia stipulaceae and has shown a spasmolytic effect in guinea pig ileum. Thus, we aimed to characterize its relaxant mechanism of action. FGAL exhibited a higher relaxant effect on ileum pre-contracted by histamine (EC50=1.9±0.4×10(-7) M) than by KCl (EC50=2.6±0.5×10(-6) M) or carbachol (EC50=1.8±0.4×10(-6) M). The flavonoid inhibited the cumulative contractions to histamine, as well as to CaCl2 in depolarizing medium nominally Ca(2+)-free. The flavonoid relaxed the ileum pre-contracted by S-(-)-Bay K8644 (EC50=9.5±1.9×10(-6) M) but less potently pre-contracted by KCl or histamine. CsCl attenuated the relaxant effect of FGAL (EC50=1.1±0.3×10(-6) M), but apamin or tetraethylammonium (1mM) had no effect (EC50=2.6±0.2×10(-7) and 1.6±0.3×10(-7) M, respectively), ruling out the involvement of small and big conductance Ca(2+)-activated K(+) channels (SKCa and BKCa, respectively). Either 4-aminopyridine or glibenclamide attenuated the relaxant effect of FGAL (EC50=1.8±0.2×10(-6) and 1.5±0.5×10(-6) M, respectively), indicating the involvement of voltage- and ATP-sensitive K(+) channels (KV and KATP, respectively). FGAL did not alter the viability of intestinal myocytes in the MTT assay and decreased (88%) Fluo-4 fluorescence, indicating a decrease in cytosolic Ca(2+) concentration. Therefore, the relaxant mechanism of FGAL involves pseudo-irreversible noncompetitive antagonism of histaminergic receptors, KV and KATP activation and blockade of CaV1, thus leading to a reduction in cytosolic Ca(2+) levels.

Blocking the L-type Ca2+ channel (Cav 1.2) is the key mechanism for the vascular relaxing effect of Pterodon spp. and its isolated diterpene methyl-6α-acetoxy-7ß-hydroxyvouacapan-17ß-oate.

Pterodon spp. Vogel (Fabaceae), popularly known as "sucupira", has ethnopharmacological application which is described as having antispasmodic and relaxant effects. Hence, it was hypothesized that sucupira oil-resin (SOR) could induce smooth muscle relaxation. So, this study investigated the mechanisms involved in the vasorelaxant effect of SOR and its isolated diterpene (methyl-6α-acetoxy-7ß-hydroxyvouacapan-17ß-oate). Vascular reactivity experiments were performed using rat aortic rings (n=5-8) with (E+) or without endothelium (E-) in an isolated bath organ. The SOR (0-56 µg/mL) relaxed phenylephrine (E+: 86.7±7.1%; E-: 92.3±4.7%) and KCl contracted rings (E-: 97.1±2.8%). In the same way, diterpene (0-48 µg/mL) also relaxed phenylephrine (E+: 94.5±3.6%; E-: 92.2±3.4%) and KCl contracted rings (E-: 99.7±0.2%). The pre-incubation of arterial rings with cyclopiazonic acid (reticular Ca2+-ATPase inhibitor), tetraethylammonium (K+ channels blocker) or MDL-12,330A (adenylyl cyclesinhibitor) did not modify either SOR- or diterpeneinduced vasorelaxation. However, ODQ (guanylyl cyclase inhibitor) impaired only diterpene-induced vasorelaxation. SOR and diterpene significantly reduced CaCl2-induced contraction stimulated by Bay K8644 (1 µM), phenylephrine (0.1 µM) or KCl solution (40 mM). Computational molecular docking studies demonstrated that the vasodilator effect of diterpene relies on blocking the Cav 1.2 channel, and patch clamp results showed that diterpene substantially decreased the ionic current through Cav 1.2 in freshly dissociated vascular smooth muscle cells. These findings suggest that SOR and its isolated diterpene induce endothelium-independent vascular relaxation by blocking the L-type Ca2+ channel (Cav 1.2).

Reduction in renal blood flow following administration of norepinephrine and phenylephrine in septic rats treated with Kir6.1 ATP-sensitive and KCa1.1 calcium-activated K+ channel blockers.

We evaluated the effects of K+ channel blockers in the vascular reactivity of in vitro perfused kidneys, as well as on the influence of vasoactive agents in the renal blood flow of rats subjected to the cecal ligation and puncture (CLP) model of sepsis. Both norepinephrine and phenylephrine had the ability to increase the vascular perfusion pressure reduced in kidneys of rats subjected to CLP at 18 h and 36 h before the experiments. The non-selective K+ channel blocker tetraethylammonium, but not the Kir6.1 blocker glibenclamide, normalized the effects of phenylephrine in kidneys from the CLP 18 h group. Systemic administration of tetraethylammonium, glibenclamide, or the KCa1.1 blocker iberiotoxin, did not change the renal blood flow in control or septic rats. Norepinephrine or phenylephrine also had no influence on the renal blood flow of septic animals, but its injection in rats from the CLP 18 h group previously treated with either glibenclamide or iberiotoxin resulted in an exacerbated reduction in the renal blood flow. These results suggest an abnormal functionality of K+ channels in the renal vascular bed in sepsis, and that the blockage of different subtypes of K+ channels may be deleterious for blood perfusion in kidneys, mainly when associated with vasoactive drugs.

Effects of flavone on the contractile activity of the circular smooth muscle of the rabbit middle colon in vitro.

The circular smooth muscles of the middle colon of the rabbit generate giant contractions of high amplitude and low frequency. Flavone, at various concentrations, reduces the giant contractions and the tonic contraction induced by 10 µM carbachol and 80 mM KCl. The contractions induced by dequalinium and tetraethylammonium are reduced by flavone (30 µM). At 100 µM, flavone decreases the contraction induced by 100 µM methylene blue and 1mM orthovanadate. These results suggest that flavone inhibit the giant contractions by (1) inhibition of voltage-dependent Ca(2+) channels, (2) activation of guanyl cyclase, (3) opening of K(+) channels and (4) inhibition of tyrosines kinases.

An analytical model of memristors in plants.

The memristor, a resistor with memory, was postulated by Chua in 1971 and the first solid-state memristor was built in 2008. Recently, we found memristors in vivo in plants. Here we propose a simple analytical model of 2 types of memristors that can be found within plants. The electrostimulation of plants by bipolar periodic waves induces electrical responses in the Aloe vera and Mimosa pudica with fingerprints of memristors. Memristive properties of the Aloe vera and Mimosa pudica are linked to the properties of voltage gated K(+) ion channels. The potassium channel blocker TEACl transform plant memristors to conventional resistors. The analytical model of a memristor with a capacitor connected in parallel exhibits different characteristic behavior at low and high frequency of applied voltage, which is the same as experimental data obtained by cyclic voltammetry in vivo.

Memory elements in the electrical network of Mimosa pudica L.

The fourth basic circuit element, a memristor, is a resistor with memory that was postulated by Chua in 1971. Here we found that memristors exist in vivo. The electrostimulation of the Mimosa pudica by bipolar sinusoidal or triangle periodic waves induce electrical responses with fingerprints of memristors. Uncouplers carbonylcyanide-3-chlorophenylhydrazone and carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone decrease the amplitude of electrical responses at low and high frequencies of bipolar sinusoidal or triangle periodic electrostimulating waves. Memristive behavior of an electrical network in the Mimosa pudica is linked to the properties of voltage gated ion channels: the channel blocker TEACl reduces the electric response to a conventional resistor. Our results demonstrate that a voltage gated K(+) channel in the excitable tissue of plants has properties of a memristor. The discovery of memristors in plants creates a new direction in the modeling and understanding of electrical phenomena in plants.

Extracellular proteolysis of reelin by tissue plasminogen activator following synaptic potentiation.

The secreted glycoprotein reelin plays an indispensable role in neuronal migration during development and in regulating adult synaptic functions. The upstream mechanisms responsible for initiating and regulating the duration and magnitude of reelin signaling are largely unknown. Here we report that reelin is cleaved between EGF-like repeats 6-7 (R6-7) by tissue plasminogen activator (tPA) under cell-free conditions. No changes were detected in the level of reelin and its fragments in the brains of tPA knockouts, implying that other unknown proteases are responsible for generating reelin fragments found constitutively in the adult brain. Induction of NMDAR-independent long-term potentiation with the potassium channel blocker tetraethylammonium chloride (TEA-Cl) led to a specific up-regulation of reelin processing at R6-7 in wild-type mice. In contrast, no changes in reelin expression and processing were observed in tPA knockouts following TEA-Cl treatment. These results demonstrate that synaptic potentiation results in tPA-dependent reelin processing and suggest that extracellular proteolysis of reelin may regulate reelin signaling in the adult brain.

Natural product vindoline stimulates insulin secretion and efficiently ameliorates glucose homeostasis in diabetic murine models.

ETHNOPHARMACOLOGICAL RELEVANCE: Catharanthus roseus (L). Don (Catharanthus roseus) is a traditional anti-diabetic herb widely used in many countries, and the alkaloids of Catharanthus roseus are considered to possess hypoglycemic ability. AIM OF THE STUDY: To systematically investigate the potential anti-diabetic effects and the underlying anti-diabetic mechanisms of vindoline, one of the alkaloids in Catharanthus roseus. MATERIALS AND METHODS: The regulation of vindoline against the glucose-stimulated insulin secretion (GSIS) was examined in insulinoma MIN6 cells and primary pancreatic islets. Insulin concentration was detected by Elisa assay. Diabetic models of db/db mice and type 2 diabetic rats induced by high-fat diet combining with streptozotocin (STZ/HFD-induced type 2 diabetic rats) were used to evaluate the anti-diabetic effect of vindoline in vivo. Daily oral treatment with vindoline (20mg/kg) to diabetic mice/rats for 4 weeks, body weight and blood glucose were determined every week, oral glucose tolerance test (OGTT) was performed after 4 weeks. RESULTS: Vindoline enhanced GSIS in both glucose- and dose-dependent manners (EC50 = 50 µM). It was determined that vindoline acted as a Kv2.1 inhibitor able to reduce the voltage-dependent outward potassium currents finally enhancing insulin secretion. It protected ß-cells from the cytokines-induced apoptosis following its inhibitory role in Kv2.1. Moreover, vindoline (20mg/kg) treatment significantly improved glucose homeostasis in db/db mice and STZ/HFD-induced type 2 diabetic rats, as reflected by its functions in increasing plasma insulin concentration, protecting the pancreatic ß-cells from damage, decreasing fasting blood glucose and glycated hemoglobin (HbA1c), improving OGTT and reducing plasma triglyceride (TG). CONCLUSION: Our findings suggested that vindoline might contribute to the anti-diabetic effects of Catharanthus roseus, and this natural product may find its more applications in the improvement of ß-cell dysfunction and further the potential treatment of type 2 diabetes.

Functional expression of choline transporter-like protein 1 (CTL1) in small cell lung carcinoma cells: a target molecule for lung cancer therapy.

Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in the small cell lung carcinoma cell line NCI-H69. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake buffer strongly enhanced choline uptake. This increase in choline uptake under the Na(+)-free conditions was inhibited by dimethylamiloride (DMA), a Na(+)/H(+) exchanger (NHE) inhibitor. Various organic cations and the choline analog hemicholinium-3 (HC-3) inhibited the choline uptake and cell viability. A correlation analysis of the potencies of organic cations for the inhibition of choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed. HC-3 and CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. The conversion of choline to ACh was confirmed, and this conversion was enhanced under Na(+)-free conditions, which in turn was sensitive to HC-3. These results indicate that choline uptake through CTL1 is used for ACh synthesis. Both an acetylcholinesterase inhibitor (eserine) and a butyrylcholinesterase inhibitor (ethopropazine) increased cell proliferation, and these effects were inhibited by 4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the choline transporter CTL1 which uses a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. This system primarily supplies choline for the synthesis of ACh and secretes ACh to act as an autocrine/paracrine growth factor, and the functional inhibition of CTL1 could promote apoptotic cell death. Identification of this new CTL1-mediated choline transport system provides a potential new target for therapeutic intervention.