Role of inducible nitric-oxide synthase in regulation of whole-cell current in lung epithelial cells.

Lung inflammation is associated with enhanced expression of proinflammatory cytokines and increased production of nitric oxide (NO) by inducible NO synthase (iNOS). To investigate the possible relationship between cytokine-induced expression of iNOS and epithelial ion channel function, we measured whole-cell current in A549 cells treated with a mixture of cytokines: tumor necrosis factor, interleukin-1 beta, and interferon-gamma for 12 h. Cytokines significantly increased the expression and activity of iNOS, and reduced generation of cGMP in response to stimulation with NO donor S-nitroso-glutathione (GSNO). Patch-clamp studies showed that 100 microM GSNO increased the whole-cell current from 11.2 +/- 1.8 to 19.6 +/- 2.7 pA/pF (n = 16) in control cells, but had no effect in cytokine-treated cells (n = 9). N-(3-(Aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of iNOS, restored activation of the current by GSNO in cytokine-treated cells, indicating a crucial role for iNOS in this process. Cells treated with cytokines showed increased levels of peroxynitrite (ONOO(-)), compared with the control, or cells that were treated with the cytokines and 1400W or superoxide dismutase/catalase. Treatment of cells with 100 microM ONOO(-) had no effect on the whole-cell current, but in contrast to untreated cells, subsequent application of GSNO did not activate the current. In conclusion, cytokine-induced expression of iNOS affects activation of the whole-cell current via NO/cGMP pathway, likely by increasing the generation of ONOO(-).

Nitric oxide activates chloride currents in human lung epithelial cells.

Epithelial Cl- channels are regulated by various physiological factors, including guanosine 3',5'-cyclic monophosphate (cGMP). Because cGMP mediates many of the physiological actions of nitric oxide (NO), we have studied both the presence of endogenous NO and the effects of exogenous NO on Cl- currents in A549 human lung epithelial cells. We have detected Ca(2+)-dependent NO synthase activity in A549 cells. Using the perforated patch-clamp technique, we have shown that inhibition of this enzyme by NG-monomethyl-L-arginine decreased Cl- current, an effect that was reversed by the NO donor S-nitrosoglutathione (GSNO). In addition, the NO donors GSNO and S-nitroso-N-acetyl-D,L-penicillamine increased whole-cell Cl- currents in A549 cells. This stimulatory effect of the NO donors was sensitive to inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, suggesting that channels other than the cystic fibrosis transmembrane conductance regulator (CFTR) are involved in the action of NO on A549 cells. In addition, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor of soluble guanylyl cyclase, decreased NO-mediated stimulation of Cl- currents. Our results suggest that, in lung epithelial cells, NO regulates a non-CFTR Cl- conductance acting via a cGMP-dependent mechanism.

Characterization and regulation of a chloride channel from bovine tracheal epithelium.

1. The patch-clamp technique was used to characterize chloride channels from the apical membranes of bovine tracheal epithelial cells. Application of GTP gamma S or NaF to excised patches revealed the existence of a novel type of Cl- channel regulated by G-proteins in a membrane-delimited manner. 2. The channel had a linear current-voltage relationship, with a conductance of 100-120 pS. Its open probability was independent of voltage. 3. The channel was highly anion selective (permeability ratio, PNa/PCl = 0.06 +/- 0.04) and had the halide permeability sequence: I- > Br- > or = Cl- > F-, corresponding to the Eisenman I sequence. This suggested that neither ionic size nor diffusion rate determined ion permeation through the channel. 4. The mole fraction behaviour was studied using fluoride and chloride ions. Mixtures of ions produced currents that would be expected from the linear combination of the two ions acting independently, indicating relatively simple permeation through the pore and compatible with a single ion binding site. 5. The channel was inhibited by the stilbene disulphonates SITS (4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulphonic acid) and DNDS (4,4'-dinitrostilbene-2,2'-sulphonic acid). SITS introduced voltage dependence to channel gating and indicated the possible involvement of lysine residues in the channel permeation pathway. 6. NaF was unable to activate Cl- channels in the presence of the aluminum chelator, deferoxamine mesylate. This indicates that Al3+ ions play an important role in chloride channel activation by fluoride. NaF activation was not dependent on the presence of calcium ions. 7. The channel was insensitive to alkaline phosphatase and to the specific inhibitors of protein phosphatase types I and 2A, okadaic acid and calyculin A. 8. The channels could be activated by GTP gamma S or by NaF in the presence of the phospholipase A2 inhibitor quinacrine, indicating that this enzyme is not involved in channel regulation.

Spinal segmental sympathetic outflow to cervical sympathetic trunk, vertebral nerve, inferior cardiac nerve and sympathetic fibres in the thoracic vagus.

The spinal segmental localization of preganglionic neurons which convey activity to the sympathetic nerves, i.e. vertebral nerve, right inferior cardiac nerve, sympathetic fibres in the thoracic vagus and cervical sympathetic trunk, was determined on the right side in chloralose anaesthetized cats. For that purpose the upper thoracic white rami were electrically stimulated with a single pulse, suprathreshold for B and C fibres, and the evoked responses were recorded in the sympathetic nerves. The relative preganglionic input from each segment of the spinal cord to the four sympathetic nerves was determined from the size of the evoked responses. It was found that each sympathetic nerve receives a maximum preganglionic input from one segment of the spinal cord (dominant segment) and that the preganglionic input gradually decreased from neighbouring segments. The spinal segmental preganglionic outflow to the cervical sympathetic trunk, thoracic vagus, right inferior cardiac nerve and vertebral nerve gradually shifted from the most rostral to the most caudal spinal cord segments. In some cases, a marked postganglionic component was found in the cervical sympathetic trunk. It was evoked by preganglionic input from the same spinal cord segments which transmitted activity to the vertebral nerve. These results indicate that there is a fixed relation between the spinal segmental localization of preganglionic neurons and the branch of the stellate ganglion receiving the input from these neurons.

Neural control of airway pressures in rabbits.

In twenty anaesthetized and spontaneously breathing rabbits airway pressures were measured above and below the larynx during tidal respiration through the larynx. Peak inspiratory and expiratory pressures at both sites were recorded in control conditions and then compared to values obtained in the course of progressive denervation of the airways. The two methods of denervation consisted of (1) bilateral section of superior and recurrent laryngeal nerves and of the midcervical vagotomy (horizontal method); (2) right-sided sections of the three nerves followed by left-sided sections (vertical method). Motor denervation of the larynx due to RLNs neurotomy (horizontal method) produced significant increases in intratracheal pressures in both phases of the respiratory cycle. Less prominent increments in pressures were achieved on RLNs neurotomy in the vertical method. SLNs section and vagotomy had little additional effect on airway pressures. Our results indicate that unilateral laryngeal palsy poses far smaller obstruction to breathing than simultaneous bilateral denervation, and that afferent denervation of the larynx has no effect on airway pressures.

Control of the heart rate by sympathetic nerves in cats.

Pre- and postganglionic sympathetic nerves were electrically stimulated and heart rate was recorded in chloralose-anaesthetised cats. The vagal nerves and white rami were cut on both sides. Electrical stimulation was performed with a 15- or 30-s train of 0.2-ms pulses at a frequency of 30 Hz. The control heart rate was 150 beats/min. Heart rate was increased when the T3 white ramus on the left (52 beats/min above control) and T3, T4 white rami on the right side (100 beats/min above control) were stimulated electrically. The magnitude of the heart rate increase declined when the neighbouring thoracic white rami were stimulated. The increase of the heart rate was caused by group B preganglionic fibres. Electrical stimulation of the sympathetic fibres in the right vagus nerve and the right inferior cardiac nerve increased the heart rate by 92 beats/min and by 67 beats/min above the control level respectively. Electrical stimulation of the left inferior cardiac nerve, the left middle cardiac nerve and the sympathetic fibres in the left vagus nerve resulted in an increase of the heart rate of 43 beats/min, 30 beats/min and 49 beats/min from the control level respectively. This indicates that a majority of the preganglionic cardiac sympathetic fibres, whose activity influences the heart rate, originate from the T3 and T4 segments of the spinal cord. The majority of the postganglionic cardiac sympathetic fibres which affect the heart rate are located in the vagal nerves.

Effect of the larynx on ventilation and respiratory pattern in anesthetized rabbits.

The effects of the larynx on ventilation and pattern of breathing have been investigated in anesthetized, spontaneously breathing rabbits. Breathing was either via a tracheostomy or via a supralaryngeal tube in control condition, after laryngeal denervation and after subsequent bilateral midcervical vagotomy. Laryngeal resistance was measured in all experimental conditions when breathing was through the larynx. In control conditions the presence of the larynx in the breathing circuit, as compared to breathing through the tracheostomy, slightly but significantly lowered inspiratory and expiratory airflows, tidal volume, and minute ventilation and increased tracheal pressure. Inspiratory and expiratory durations were not significantly changed. Expiratory laryngeal resistance was higher than inspiratory. Laryngeal deafferentation did not significantly modify values of the respiratory variables. Subsequent motor denervation of the larynx enhanced the decrease in ventilatory parameters due to adding the larynx to the circuit and lengthened the respiratory cycle. Inspiratory laryngeal resistance increased sevenfold and expiratory resistance threefold. Subsequent midcervical vagotomy induced a further increase in inspiratory and expiratory durations and augmented tidal volume independent of the route of breathing, and also reduced laryngeal resistance previously increased by motor denervation. These results reveal the ventilatory effects of the larynx and show the importance of its patency in the pattern of breathing.

Phrenic output changes due to progressive airway denervation in rabbits.

The experiments were performed with thirteen anaesthetized rabbits breathing spontaneously through the larynx. Phrenic output was measured during tidal respiration in control conditions and in the course of progressive denervation of the laryngeal and tracheobronchial compartments. We have analysed changes in amplitude and the rate of rise of the integrated phrenic neurogram as well as the changes in TI, TE, TT and f of the respiratory timing after superior laryngeal nerves (SLN-s) section, section of the recurrent laryngeal nerves (RLN-s) and step-wise complete midcervical vagotomy. Nerve sections were performed by two methods: 1) bilateral SLN-s, RLN-s and cervical vagal neurotomy (horizontal method); 2) right-sided neurotomies of SLN, RLN and vagus followed by left-sided neurotomies of these nerves (vertical method). Laryngeal deafferentation did not greatly affect respiratory variables. Unilateral and especially bilateral vocal cord paralysis prolonged the respiratory cycle with enhanced TI/TT ratio, increased amplitude and rate of rise of the integrated phrenic neurogram. Maximum values of all parameters (the rate of rise excluded) were achieved after complete midcervical vagotomy. Of the two methods of denervation, the right-sided followed by left-sided denervation led to better adaptation of the organism to the respiratory disturbance (smaller changes in TI and TE compared with the control values). Denervation by the 'horizontal' method (paired section of the nerves) led to an abrupt failure of effective ventilation.