Most exhaled water is produced as gaseous water vapor, which can be collected in cooled condensers. The presence of nonvolatile solutes in these condensates suggests that droplets of respiratory fluid (RF) have also been collected. However, calculation of RF solute concentrations from condensates requires estimation of the dilution of RF droplets by water vapor. We used condensate electrolyte concentrations to calculate the dilution of RF droplets in condensates from 20 normal subjects. The total ionic concentration (conductivity) was 497 plus minus 68 (mean plus minus SEM) muM. Of this, 229 plus minus 43 muM was NH(4)(+), but little NH(4)(+) was collected from subjects with tracheostomies, indicating oral formation. The Na+ concentration in condensate ([Na+](cond)) averaged 242 plus minus 43 muM. Large variations in [Na(+)](cond) correlated well with variations of K+ in condensate ([K+](cond)) and Cl-) in condensate ([Cl-](cond)), and were attributed to differences in respiratory droplet dilution. Dividing condensate values of ([Na+] + [K+] ) by those of plasma indicated that RF represented between 0.01% and 2.00% of condensate volumes. Calculated values for Na+, K+, Cl-, lactate, and protein in RF were [Na+](RF) = 91 +/- 8 mM, [K+](RF) = 60 +/- 11 mM, [Cl-](RF) = 102 +/- 17 mM, [lactate](RF) = 44 +/- 17 mM, and [protein](RF) = 7.63 +/- 1.82 g/dl, respectively.
Exhaled breath condensates have been widely used to detect inflammatory mediators in the fluid that covers airway surfaces of patients with inflammatory lung disorders. This approach is much less invasive than bronchoalveolar lavage, but respiratory droplets are markedly diluted by large and variable amounts of water vapor. We estimated the dilution of respiratory droplets by comparing concentrations of nonvolatile, reference indicators (total nonvolatile cations, urea or conductivity) in 18 normal subjects with normal plasma concentrations by assuming similar concentrations in the respiratory fluid and plasma. The volatile cation, NH4+ (most of which is delivered as NH3 gas from the mouth), represented 93 +/- 3% (SEM) of the condensate cations. More than 99% of the NH4+ was removed by lyophilization, making it possible to use conductivity to estimate total nonvolatile ionic concentrations and facilitating analysis of urea. Conductivity was significantly correlated with electrolyte and urea concentrations. Estimates of dilution based on total cations, conductivity, and urea were not significantly different (cations: 20,472 +/- 2,516; conductivity: 21,019 +/- 2,427; and urea: 18,818 +/- 2,402). These observations suggest that the conductivity of lyophilized samples can be used as an inexpensive, simple, and reliable method for estimating dilution of nonvolatile, hydrophilic mediators in condensates.
Discrete, localized elevations of myoplasmic [Ca2+], Ca2+ 'sparks', were readily detected using the fluorescent Ca2+ indicator fluo-3 and laser scanning confocal microscopy in 'dyspedic' 1B5 myotubes, i.e. myotubes which do not express ryanodine receptors (RyRs), transduced with virions containing cDNA for RyR type 3 that were saponin permeabilized to allow dye entry. Ca2+ sparks were never observed in non-transduced RyR null myotubes. The spatial locations of sparks observed in permeabilized myotubes roughly corresponded to regions of RyR protein expression in the same myotube as detected after subsequent fixation and antibody staining. Permeabilized RyR3-transduced myotubes exhibited similar punctate peripheral RyR3 protein immunohistochemical patterns as myotubes fixed before permeabilization indicating that permeabilization did not affect the structural organization of the triad. Ca2+ sparks, recorded in line scan mode, in permeabilized myotubes expressing RyR3 exhibited mean amplitudes (change in fluorescence/mean fluorescence, DeltaF/F: 1.20 +/- 0.04) and temporal rise times (10-90%; 6.31 +/- 0.12 ms) similar to those of sparks recorded in permeabilized frog skeletal muscle fibres (0.98 +/- 0.01; 6.11 +/- 0.07, respectively) using the same confocal system. Spatial extent and temporal duration of the Ca2+ sparks were approximately 40% larger in the RyR3-expressing myotube cultures than in frog fibres. Ca2+ sparks recorded in line scan mode often occurred repetitively at the same spatial location in RyR3-expressing myotubes. Such repetitive events were highly reproducible in amplitude and spatio-temporal properties, as previously observed for repetitive mode sparks in frog skeletal muscle. Ca2+ sparks recorded in xy mode were frequently compressed in the y (slower scan) direction compared to the x direction. This asymmetry was reproduced assuming spatially symmetric events having the time course of Ca2+ sparks recorded in line scan (xt) mode. These expression studies demonstrate that the presence of RyR3 is sufficient for the production of Ca2+ sparks in a skeletal muscle system lacking the expression of any other RyR isoform.
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