Activation of chloride transport in CF airway epithelial cell lines and primary CF nasal epithelial cells by S-nitrosoglutathione.

BACKGROUND: It has been suggested that low microM concentrations of S-nitrosoglutathione (GSNO), an endogenous bronchodilator, may promote maturation of the defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR). Because nitric oxide (NO) and GSNO levels appear to be low in the CF airway, there is an interest in the possibility that GSNO replacement could be of therapeutic benefit in CF. METHODS: The effect of GSNO on chloride (Cl-) transport was investigated in primary nasal epithelial cells obtained from CF patients homozygous for the delF508 mutation, as well as in two CF cell lines (CFBE and CFSME), using both a fluorescent Cl- indicator and X-ray microanalysis. Maturation of delF508 CFTR was determined by immunoblotting. RESULTS: Treatment with 60 microM GSNO for 4 hours increased cAMP-induced chloride efflux in nasal epithelial cells from 18 out of 21 CF patients, but did not significantly affect Cl- efflux in cells from healthy controls. This Cl- efflux was confirmed by measurements with a fluorescent Cl- indicator in the CFBE and CFSME cell lines. The effect of GSNO on Cl- efflux in CFBE cells could be inhibited both by a specific thiazolidinone CFTR inhibitor (CFTRinh-172) and by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS). X-ray microanalysis showed that, following 4 hours incubation with 60 microM GSNO, cAMP agonists caused a decrease in the cellular Cl- concentration in CFBE cells, corresponding to Cl- efflux. GSNO exposure resulted in an increase in the protein expression and maturation, as shown by immunoblot analysis. GSNO did not increase the cytosolic Ca2+ concentration in cultured airway epithelial cells. CONCLUSION: Previous studies have suggested that treatment with GSNO promotes maturation of delF508-CFTR, consistent with our results in this study. Here we show that GSNO increases chloride efflux, both in the two CF cell lines and in primary nasal epithelial cells from delF508-CF patients. This effect is at least in part mediated by CFTR. GSNO may be a candidate for pharmacological treatment of the defective chloride transport in CF epithelial cells.

PC12 and HEL cell communication in a reconstituted synapse analyzed with mathematical modeling.

A synapse simulating model comprising of the nerve growth factor (NGF)-differentiated PC12 cells releasing neurotransmitter (NT) and sensor 92.1.7.human erythroleukemia (HEL) cells has been used for simulating the connection between neurons and target cells. A Ca(2+) elevation was observed in both cell types when the PC12 cells were challenged with nicotine. The response patterns of individual cell were subsequently analyzed mathematically. The Ca(2+) signals of the PC12 cells were described by an equation representing a simple bi-exponential function. The NT-noradrenaline discharged by the PC12 cells in response to nicotine caused heterogeneous secondary Ca(2+) elevations in the HEL cells after a certain delay. Model fitting of this response disclosed slow "hidden" oscillations and heterogeneous secondary Ca(2+) signals could be grouped on the basis of the oscillation frequency. As determined in control experiments with noradrenaline (NA), the value of oscillation frequency also revealed a good correlation with the NT concentration.

Ca2+-dependent potentiation of muscarinic receptor-mediated Ca2+ elevation.

Muscarinic receptor-mediated increases in Ca(2+) in SH-SY5Y neuroblastoma cells consist of an initial fast and transient phase followed by a sustained phase. Activation of voltage-gated Ca(2+) channels prior to muscarinic stimulation resulted in a several-fold potentiation of the fast phase. Unlike the muscarinic response under control conditions, this potentiated elevation of intracellular Ca(2+) was to a large extent dependent on extracellular Ca(2+). In potentiated cells, muscarinic stimulation also activated a rapid Mn(2+) entry. By using known organic and inorganic blockers of cation channels, this influx pathway was easily separated from the known Ca(2+) influx pathways, the store-operated pathway and the voltage-gated Ca(2+) channels. In addition to the Ca(2+) influx, both IP(3) production and Ca(2+) release were also enhanced during the potentiated response. The results suggest that a small increase in intracellular Ca(2+) amplifies the muscarinic Ca(2+) response at several stages, most notably by unravelling an apparently novel receptor-activated influx pathway.

Phospholipase C activator m-3M3FBS affects Ca2+ homeostasis independently of phospholipase C activation.

In this study, we have investigated responses to the phospholipase C (PLC) activator m-3M3FBS in SH-SY5Y human neuroblastoma cells. As measured using fura-2, m-3M3FBS caused a slowly developing - full response was obtained within 4-6 min - Ca(2+) elevation both in the presence and absence of extracellular Ca(2+), indicating Ca(2+) release from intracellular stores, putatively from endoplasmic reticulum and mitochondria. PLC activity was also measured using two methods, the classical ion-exchange separation and the more novel fluorescent real-time method. In the time frame in which m-3M3FBS caused Ca(2+) elevation (up to 7 min), no PLC activation was detected. Instead, more than 20 min were required to see any inositol phosphate generation in response to m-3M3FBS. m-3M3FBS also interfered with store-operated Ca(2+) influx and Ca(2+) extrusion. In conclusion, m-3M3FBS cannot be considered either potent or specific PLC activator.

Mechanisms of nicotine mediated communication between NGF-differentiated PC12 and HEL cells.

HEL 92.1.7 cells were immobilized among nerve growth factor (NGF)-differentiated PC12 cells. Nicotine caused an immediate Ca2+ mobilisation in the PC12 cells followed by a delayed secondary Ca2+ response in the HEL 92.1.7 cells. The Ca2+ elevation in response to nicotine in PC12 cells was abolished by Na+ removal. The response was diminished by omega-contoxin GVIA (omega-CTx-GVIA) in PC12 cell neurites and by nifedipine in the cell bodies, respectively. The secondary response in HEL 92.1.7 cells was blocked by omega-CTx-GVIA. The results suggest that nicotinic receptor-mediated depolarisation and subsequent activation of voltage dependent Ca2+ channels (VDCC) are sufficient to induce transmitter release from NGF-differentiated PC12 cell varicosities without requirement for additional Ca2+ influx via nicotinic receptor ion channels.