Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisors: Clifford Keil and Vincent D'Amico, Dept. of Entomology & Wildlife Ecology. Includes bibliographical references.

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

beta-Bungarotoxin (beta-BuTx), the presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide subunits. A phospholipase A2 subunit named A chain, and a non-phospholipase A2 subunits named B chain. The A chain and B chain are covalently linked by one disulfide bridge. Although it has been widely accepted that the toxic effect of beta-BuTx is attributed to the disturbance of presynaptic transmitter release, however the inhibition of transmitter release by beta-BuTx is still obscure. Here we investigate the mechanism that mediates facilitation of transmitter release at the neuromuscular junction induced by beta-BuTx, using Xenopus nerve-muscle coculture. Application of beta-BuTx and isotoxins BM12, BM13 led to a marked increase in the frequency of spontaneous synaptic currents (SSCs) after a short period (12~18 min) of latency. The synaptic potentiation induced by these toxins was abolished when Ca2+ in the medium is substituted by Ba2+ (a potent phospholipase A2 inhibitor). Application of PLP-BM12 and PLP-BM13, which have been chemical-modification to lose their PLA2 activity from BM12 and BM13, failed to potentiate the transmitter release.

Reproduction de : Thèse de doctorat : Biologie cellulaire et moléculaire : Lyon 1 : 2004. / Titre provenant de l'écran titre. 256 réf. bibliogr.

Zugl.: Giessen, Univ., Diss., 2007

Adiponectin (ADP) is an adipokine, which acts as an insulin sensitizing hormone. Recent studies have shown that adiponectin receptors (AdipoR1, AdipoR2) are present in the CNS; however, there is some debate as to whether or not ADP crosses the blood brain barrier (BBB). Circumventricular organs (CVO) are CNS sites outside the BBB, and thus represent sites at which circulating adiponectin may act to influence the CNS without having to cross the BBB. The subfornical organ (SFO) is a CVO that is responsive to a number of different circulating satiety signals including amylin, CCK, and ghrelin. We report here that the SFO also shows a high density of mRNA for both adiponectin receptors. These observations support the concept that the SFO may be a key player in sensing circulating ADP. To test the hypothesis that ADP influences the excitability of SFO neurons, we used current-clamp electrophysiology on dissociated SFO neurons to observe changes in membrane potential. ADP (10 nM) application effected the excitability of SFO neurons, where the cells either depolarized (8.9±0.9 mV, 21 of 97 cells) or hyperpolarized (-8.0±0.5 mV, 34 of 97 cells). Using single-cell RT-PCR we found that the majority of the responsive neurons expressed AdipoR1 or R2 and the non-responsive neurons expressed neither. In view of the recognized role of ADP in the regulation of energy balance, we next examined the effects of food deprivation for 48 hours on ADP signaling in the SFO. Our previous microarray analysis of SFO showed increases in AdipoR2 mRNA, with no significant change in AdipoR1 mRNA. We have also assessed the effects of such changes in receptor expression on ADP signaling in SFO neurons using calcium imaging and patch clamp techniques. In SFO neurons obtained from control animals, ADP induced increases in intracellular Ca2+ were observed in 25% of cells, while following food deprivation 0% of cells showed this response. Furthermore, 77% of neurons from starved animals showed clear depolarization, while no hyperpolarizing responses were observed. The results presented in this study suggest that adiponectin modulates the excitability of SFO neurons and that the response to ADP changes during starvation. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2008-09-17 18:07:35.099

Universiẗat, Diss., 2007--Giessen.

Zugl.: Giessen, Univ., Diss., 2007

Universiẗat, Diss.--München, 1995.