Fluorescent Studies On The Modulation Of Cytosolic Calcium Signaling Of Lymphocytes By Little Extrinsic Molecules

Chapter 1 The modulation of cytosolic calcium signaling of lymphocytes was studied by little extrinsic molecules,useing fura-2 and fluo-3 as fluorescence probe.Ca²⁺ as a universal intracellular messenger and regulates a broad of physiological functions.The specific and coordinated activation of many cellular processes by stimulus-induced changes in the intracellular Ca²⁺ concentration[Ca²⁺]_i requires appropriate temporal and spatial coding patterns.As a result,low agonist concentrations cause Ca²⁺ oscillations or repetitive Ca²⁺ spikes arising from cyclical Ca²⁺ release from,and Ca²⁺ reuptake into,the endoplasmic reticulum,whereby agonist concentration and in the frequency of Ca²⁺ osillations arecorrelated.Thus,the Ca²⁺ signal is frequency-modulated.The aim of this study was to investigate the molecular mechanisms responsible for the generation of these amplitude-modulated Ca²⁺ signal.Chapter 2 The effect of penicillin on intracellular free calcium concentration was studied by monitoring the fluorescence of human peripheral lymphocytes loaded with fura-2.High internal Na⁺ was expected to increase the driving force for Ca²⁺ influx via Na⁺/Ca²⁺ exchanger,and under the effect of penicillin high internal Na⁺ was also able to increase the driving force for Ca²⁺ efflux.Furthermore,when penicillin acted on the cells, a substantial role for the Ca²⁺ efflux through a Na⁺-dependent mode in Ca_i²⁺ homeostasis was indicated.Decreased temperature significantly slowed the Ca²⁺ elevation in Na⁺-PSS solution,it seemed that penicillin had little influence on the increasing of[Ca²⁺]_i induced by higher temperature.The effects of penicillin G were retained in the presence of nikadipine,a potent inhibitor of voltage-dependent calcium influx pathways.These results showed that penicillin G stimulated Ca²⁺ extrusion following Na⁺-dependent transporters. Both an increase in cytosolic[Ca²⁺]and reversal of the Na⁺ gradient are necessary to demonstrate enhanced effect of penicillin on Ca²⁺ homeostasis.The more[Ca²⁺]_i increased, the more penicillin effected.In conclusion,penicillin-induced decreases in intracellular free calcium concentrations and stimulated Ca²⁺ extrusion following Na⁺-dependent transporters.It is evident that penicillin can induce the Ca²⁺ release,which can be produced independent of ouabain.The results support the conclusion of the penicillin effect studies and indicate that there is involment of the Na⁺/Ca²⁺ antiporter system and/or calcium channels in the Ca²⁺ mobilization induced by reversal Na⁺ gradient in lymphocytes.The general increase trend of[Ca²⁺]_i can be diminished by penicillin.Chapter 3 Cefoperazone is widely distributed into most body fluids and tissues reaching conc- entrations higher than the minimum concentrations of susceptible bacteria.We first investigated the effect of cefoperazone on the resting[Ca²⁺]_i of human lymphocytes. With the increasing concentration of cefoperazone,the remarkable decreases of[Ca²⁺]_i were observed.The basal[Ca²⁺]_i in resting human peripheral lymphocytes was 100±10nM.In a Ca²⁺-containing solution,addition of 200μg/mL cefoperazone to the extracellular medium produced a cytosolic calcium decrease of 55±11 nmol/L. However,this calcium decrease was observed as 20±11 nmol/L in a Ca²⁺-free solution, suggesting that,it might be due to the inhibition of calcium influx from the external medium³.The more[Ca²⁺]_i increased,the more cefoperazone effected.In a Na⁺-free and Ca²⁺-free medium,200μg/mL cefoperazone significantly reduced a 30%decrease of [Ca²⁺]_i on control samples.These further reveal that cefoperazone induced the decline of Ca²⁺ rise in human lymphocytes by the inhibition of Na⁺-dependent Ca²⁺ entry. Preincubation with 0.1 mmol/L ouabain can operate the Na⁺/Ca²⁺ exchanger in the reverse mode by which outer Ca²⁺ can quickly enter the lymphocytes,but it had almost no effect on the cefoperazone-induced calcium flowing out.It indicated that ouabain-induced calcium entry by the reverse mode of Na⁺/Ca²⁺ exchanger was inhibited.The sustained Ca²⁺ signal relies on the operation of the ion channels in lymphocytes,hence the Ca²⁺ channels for Ca²⁺ influx are to serve as targets for this cephalosporins antibiotic,and further interactions lead to the losing of Ca²⁺ reserve in the lymphocytes.Chapter 4 Acrylamide is a small,water soluble,organic molecule being a vinyl monomer formed from the hydration of acrylonitrile.Acrylamide can increase cytosolic free Ca²⁺ concentration,but no studies have investigated the mechanism underlying acrylamide-induced increase in[Ca²⁺]_i in immune cells.In view of this,we investigated the effect of acrylamide(5-50μM) on[Ca²⁺]_i in human lymphocytes using a fluorescence Ca²⁺ indicator,fluo-3.Acrylamide caused sustained increases in[Ca²⁺]_i,in a concentration-dependent manner.The acrylamide-induced increase in[Ca²⁺]_i was abolished by the omission of extracellular Ca²⁺,suggesting that acrylamide induce a calcium influx by the opening of Ca²⁺ channels.We further employed an unspecific inhibitor of plasma membrane Ca²⁺ channels.We observed that Ni²⁺ curtailed significantly the calcium rise.The omission of extracellular Na⁺ failed to inhibit the acrylamide-induced increases in[Ca²⁺]_i.Furthermore,nicardipine had no effect on the increases in[Ca²⁺]i.but this increases was partially inhibited byω-conotoxin.Our results confirmed that acrylamide induced plasma membrane depolarization,known to be involved in the opening of the voltage-gated calcium channels.Acrylamide also induced Mn²⁺ influx into lymphocytes.These results suggest that acrylamide transiently increases [Ca²⁺]_i in lymphocytes by stimulating Ca²⁺ entry via modulating the permeability of calcium channels.Some dietary antioxidants such as Vitamin C and E can be considered as protective agents against interference action of acrylamide.Chapter 5 A fast and cost-effective method using HPLC/UV has been developed for determination of acrylamide in deep-fried flour-based leaven dough foods available in Hong Kong.The samples were purified by a simple solid-phase extraction method which combined Oasis HLB and Bond Elut-Accucat cartridges.The aqueous sample solution was centrifuged at 13000×g and 0℃for 15 rain to successfully remove the fat in the samples.A gradient elution program and a mobile phase of 4.0%(v/v) acetonitrile in water allowed sufficient retention and well resolved acrylamide from the food matrices in the sample extracts.Acrylamide was detected at UV wavelengths of 210 and 225 nm.The amounts of acrylamide in eight food samples were 27-198μg/kg when 1-g samples were analyzed.The recoveries of acrylamide were larger than 78.0%and the precisions were 2.1-10.9%(n=3),respectively.Our proposed method is especially relevant for analyzing acrylamide in those oily food matrices.