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Biomedical subjects

R C Boucher

Publications and source records attributed to R C Boucher.

At least 217 records · Page 12Linked to original sources
PubMed1988

Development, organization, and function of tight junctional complexes in the tracheal epithelium of infant ferrets.

The surface epithelium of newborn ferret airways matures rapidly in the first month of life. Prominent developmental features include a transition from predominantly non-ciliated to ciliated cells, quantitative and qualitative changes in secretion of macromolecules, and a transition from secretory to absorptive patterns of ion transport. Freeze-fracture replicas of ferret tracheal epithelium from 0 to 28 days of age exhibited progressive developmental patterns in tight junctional structure from beaded, unclosed patterns in newborns to more closed patterns at 28 days. Strand number increased while the depth of tight junctional structures and the proportion of strands exhibiting discontinuity decreased postnatally. Total transepithelial conductance, paracellular conductance, and cell size decreased over the first month. Our data suggest that changes in physiological parameters that reflect epithelial tight junction permeability can be attributed, at least in part, to maturation of this intercellular junction during the postnatal period.

Animals↗
PubMed1988

3. Epithelial injury. Mechanisms and cell biology of airway epithelial injury.

Injury of airway epithelia involves selective interactions of toxins with the cellular and paracellular components of the epithelial barrier. Repair of epithelial injury appears to involve mitogenic and differentiating factors that function in autocrine and paracrine modes. Fruitful techniques for future study of airway epithelial injury and repair will include combinations of cell culture and modulation of gene expression.

Cell Division↗
PubMed1988

Cell calcium levels of normal and cystic fibrosis nasal epithelium.

To determine whether epithelial ion transport abnormalities in cystic fibrosis (CF) might reflect abnormal regulation of intracellular Ca2+ levels, cytosolic free calcium (Cai2+) was measured using fura-2 or quin2 in suspensions of normal or CF nasal epithelial cells derived from primary cell culture. The basal Cai2+ level measured with fura-2 in CF nasal epithelia was 155 +/- 9 nM (n = 5), a value not significantly different from normal nasal epithelia (143 +/- 16 nM, n = 5). Total cell calcium was measured by atomic absorption spectroscopy and no differences were observed between CF (6.3 +/- 0.5 nmol/mg protein; n = 3) and normal (6.2 +/- 1.2 nmol/mg protein; n = 3) nasal epithelial cells. Placing Na+ loaded cells in a low (10 mM) extracellular Na+ solution resulted in a rapid increase in Cai2+ consistent with Ca2+ uptake via a plasmalemmal Na+-Ca2+ exchanger. The level of Cai2+ achieved by this low Na+ maneuver was not significantly different in CF cells compared to normal cells. Neither isoproterenol (10(-5) M) nor forskolin (10(-6) M) had any effect on Cai2+ in normal or CF nasal epithelial cells. Thus, it appears that differences in cell Cai2+, as measured by fluorescent chelators in suspensions of cultured cells, do not account for the abnormalities in basal or isoproterenol stimulated ion transport in CF tissues.

Adolescent↗
PubMed1987

Acute cardiovascular and pulmonary effects of intravenous and aerosolized amiloride in the dog.

Active Na+ absorption by the epithelia that line the airways can drive volume from the airway surface. Exposure of the lumen-facing surface of airway epithelia to amiloride inhibits Na+ transport. Consequently, aerosolized amiloride may help hydrate the desiccated surface liquid that characterizes lung diseases such as cystic fibrosis. We studied the acute cardiovascular and pulmonary effects of amiloride in anesthetized dogs. Intravenous amiloride reduced blood pressure and increased ventilation frequency and airways resistance of the dog. The effects were dose-related (ED50 = 1.3 X 10(-6) mol/kg), resembled those of injected histamine, and were antagonized by methapyrilene. These results were compatible with the release of endogenous histamine by amiloride, but the graded dose-effect relationship for amiloride differed from the quantal relationship induced by the prototypical releaser, 48/80. Aerosolization of a Ringer's solution that was nearly saturated with amiloride deposited, in upper airway surface liquid, drug concentrations which in previous studies, were sufficient to inhibit Na+ transport by canine airways (ED50 approximately 10(-6) M). Cardiovascular function, airways resistance, and indices of pulmonary tissue water, gas exchange, and perfusion were not affected. Since circulating amiloride after aerosolization was estimated to fall below the concentration that induced vasodepression and correct circled bronchoconstriction after intravenous injection, we conclude that the potency of aerosolized amiloride to induce acute effects in tissues other than airway epithelia is no greater than that of intravenous drug.

Administration, Inhalation↗
PubMed1987

Ion transport by rabbit nonciliated bronchiolar epithelial cells (Clara cells) in culture.

The functions of epithelia that line small airways in mammalian lungs are unknown. To gain insight into the role of small-airway epithelia in lung liquid balance, Clara cells were isolated from excised rabbit lungs by enzymatic digestion, enriched by centrifugal elutriation and density centrifugation, and further purified by differential adherence to collagen matrices. The resulting cell population was composed of 85% Clara cells, 3% ciliated cells, and less than 1% macrophages. The remainder of the cells were not definitively identified. The transepithelial potential difference peaked on day 3 in culture. Preparations studied in Ussing flux chambers exhibited a potential difference of 8 mV (apical bath negative), a resistance of 500 ohm X cm2, and an equivalent short-circuit current (ISCeq) of 16 microA/cm2. Inhibition of the Na+/K+-ATPase by ouabain abolished ISCeq. Exposure of the apical surface to amiloride or replacement of Na+ in the apical bathing solution with an impermeant cation (N-methyl-D-glucamine) decreased ISCeq by 66% and 93%, respectively. Neither amiloride in the basolateral bathing solution, nor bumetanide, nor isoproterenol significantly altered basal ISCeq. These findings indicate that Clara cells in culture form polarized monolayers, Clara cells transport Na+ from the apical to the basolateral bathing solution, and the small airways of the rabbit may function in liquid absorption.

Amiloride↗
PubMed1987

Effects of raised osmolarity on canine tracheal epithelial ion transport function.

Evaporation of water from upper airway surfaces increases surface liquid osmolarity. We studied the effects of raised osmolarity of the solution bathing the luminal surface of excised canine tracheal epithelium. Osmolarity was increased by adding NaCl or mannitol. NaCl addition induced a concentration-dependent fall in short-circuit current and a rise in transepithelial conductance (-33% and +14% per 100 mosM, respectively). Unidirectional isotopic fluxes of 22Na, 36Cl, and [14C]mannitol were measured in short-circuited tissues in the base-line state and after addition of NaCl or mannitol to an isotonic mucosal solution. NaCl addition (75 mM) caused a 50% increase in conductance (G) and a parallel increase in [14C]mannitol permeability (Pmann), indicating an increase in paracellular permeability. Net Cl- secretion was reduced 50%, and net Na+ absorption was unchanged despite an increased chemical gradient for absorption, indicating an inhibition of active ion transport. Mannitol addition (150 mM) abolished net Na+ absorption but did not increase G or Pmann or change net Cl- secretion. These results suggest that responses to increased tracheal surface liquid osmolarity during spontaneous breathing may occur in both the cellular (inhibition of active Na+ and Cl- transport) and paracellular (increased [14C]mannitol permeability) compartments of the mucosa.

Animals↗
PubMed1987

Abnormal apical cell membrane in cystic fibrosis respiratory epithelium. An in vitro electrophysiologic analysis.

The transepithelial chloride permeability of airway and sweat ductal epithelium has been reported to be decreased in patients with cystic fibrosis (CF). In the present study, we investigated whether the airway epithelial defect was in the cell path by characterizing the relative ion permeabilities of the apical membrane of respiratory epithelial cells from CF and normal subjects. Membrane electric potential difference (PD) and the responses to luminal Cl- replacement, isoproterenol, and amiloride were measured with intracellular microelectrodes. The PD across the apical barrier was smaller for CF (-11 mV) than normal (-29 mV) epithelia whereas the PD across the basolateral barrier was similar, (-26 and -34 mV respectively). In contrast to normal nasal epithelium, the apical membrane in CF epithelia was not Cl- permselective and was not responsive to isoproterenol. Amiloride, a selective Na+ channel blocker, induced a larger apical membrane hyperpolarization and a greater increase in transepithelial resistance in CF epithelia. Both reduced apical cell membrane Cl- conductance and increased Na+ conductance appear to contribute to the abnormal function of respiratory epithelia of CF patients.

Amiloride↗
PubMed1987

Cell culture approaches to the investigation of human airway ion transport.

Several techniques for the study of human airway epithelial ion transport employing cultured cells were explored. Human nasal epithelial cells were cultured in serum-free, hormone supplemented media. For bioelectric characterization of ion transport functions of cultured cells, cells were inoculated into heterologous grafts, and implanted into immunocompromised mice, or onto collagen membranes maintained in vitro. Cells populating either preparation exhibited a pattern of Na+ and Cl- transport similar to that of freshly excised nasal specimens. Differences between preparations were observed for absolute transport rates, tissue resistance, and morphology. We conclude that (1) cell culture techniques will be useful in investigating ion transport activities of human pulmonary epithelia from normal and abnormal lungs; and (2) the selection of specific culture techniques should be guided by the nature of the epithelial functions tested.

Adolescent↗
PubMed1986

Arachidonic acid metabolism by canine tracheal epithelial cells. Product formation and relationship to chloride secretion.

Canine tracheal epithelial cells freshly isolated from mongrel dog trachea were used to study relationships between arachidonic acid metabolism and chloride ion movement. High performance liquid chromatography (HPLC) analysis of the cell incubation media after the addition of A23187 showed the presence of prostaglandin H synthase and lipoxygenase-derived metabolites. The major prostaglandin H synthase metabolite identified by HPLC, gas chromatography, and mass spectrometry was prostaglandin (PG) D2. The major lipoxygenase metabolites were leukotriene (LT) C4 and LTB4. LTB4 was identified by HPLC, UV spectroscopy, and gas chromatography. Straight phase HPLC of the methyl esters indicated only a minor formation of LTB4 isomers. LTC4 was identified by HPLC, UV spectroscopy, and conversion to LTD4 by gamma-glutamyl transpeptidase. Analysis by radioimmunoassays indicated approximately 1-2 ng of LTB4 and peptide LT formed by 10(6) cells after A23187 stimulation. The addition of ionophore A23187 caused a rapid release of arachidonic acid metabolites which was completed within 5 min of stimulation. Cl- secretion was measured in parallel studies of excised tracheas in Ussing chambers. Cl- secretion occurred at 2-3 min after the addition of ionophore, and the most rapid change occurred with the highest PGD2 concentrations. Indomethacin produced a concentration-dependent inhibition of PGD2 formation and Cl- movement. The addition of PGE2, PGD2, and PGH2 effectively stimulated Cl- secretion. LTC4 also stimulated Cl- secretion, but the stimulation was inhibited by indomethacin. These results indicate that canine tracheal epithelial cells metabolize arachidonic acid via both prostaglandin H synthase and lipoxygenase enzymes. It appears that endogenous PGD2 formation is the important variable controlling the Cl- ion movement in canine trachea.

Animals↗
PubMed1986

Effects of formaldehyde on bronchial ion transport.

The functions of the epithelium that lines mammalian airways are potential targets for the toxic effects of reactive chemicals such as formaldehyde. We examined the effects of formaldehyde on bioelectric properties and ion permeation of excised canine and human bronchial epithelium. Concentration-dependent reductions in short-circuit current were induced in both tissues. Sodium absorption and transcellular chloride fluxes across canine bronchial epithelium were inhibited by 65 and 35%, respectively, by 10(-3) M formaldehyde. The QO2 of isolated dog bronchial epithelial cells was reduced by 32% by 10(-3) M formaldehyde. These results are consistent with an action of formaldehyde on cellular ion permeability but effects secondary to metabolic inhibition cannot be dismissed.

Animals↗
PubMed1986

Increased nasal potential difference and amiloride sensitivity in neonates with cystic fibrosis.

Patients with cystic fibrosis (CF) have an increased nasal transepithelial potential difference (PD) which reflects increased sodium absorption across epithelium relatively impermeable to chloride. To evaluate nasal epithelial function in neonates with CF, the PD was recorded and the voltage response to superfusion of 10(-5M) amiloride, an inhibitor of sodium transport, measured between a Ringer perfused bridge on the nasal mucosa and a reference electrode in the subcutaneous space. We studied three neonates with CF with meconium ileus and compared the results with those in 24 term healthy neonates, including one obligate heterozygote for CF, and 27 control neonates with disease. All three CF neonates had raised sweat chloride values (mean 100 mEq/L) at 2 months. The CF neonates had higher PDs (-64.0 +/- 8.4 mV) than those in normal (-24.4 +/- 2.0 mV) or control (-25.8 +/- 2.0 mV) neonates. Superfusion with amiloride induced a 72% reduction in PD in the CF neonates as compared with healthy (37.5 +/- 1.0%) and diseased (36.0 +/- 1.3%) neonates. The PD and amiloride response in CF neonates are similar to those in CF infants (2.24 months), older CF children (greater than 6 years), and CF adults (-64.9 +/- 9.3 mV; 77.7 +/- 1.8%, n = 51). These results suggest that (1) nasal epithelial dysfunction is present in patients with CF shortly after birth, and (2) the nasal PD may be a diagnostic adjunct to the sweat test in the early diagnosis of CF.

Adult↗
PubMed1986

Effects of Pseudomonas aeruginosa on bronchial epithelial ion transport.

During chronic lung infections the effects of bacterial exoproducts on the function of the airways epithelium could play a role in the overall pathophysiology of infection. We measured the effects of Pseudomonas aeruginosa culture on ion transport and solute permeability of dog bronchial epithelium to determine if this organism produces toxins that affect these epithelial functions. Thirty-six-hour trypticase soy broth cultures of P. aeruginosa at 1:40 dilutions decreased short-circuit current and active sodium absorption and lowered conductance and chloride unidirectional fluxes. At 1:4 dilutions, P. aeruginosa increased conductance and the permeability coefficient of 14C-mannitol, suggesting that solute flow around cells was increased. These effects appear to be caused by a heat-stable glycolipid hemolysin produced by P. aeruginosa.

Animals↗
PubMed1986

Deposition, clearance, and effects of aerosolized amiloride in sheep airways.

Persistent Na+ absorption across a relatively Cl- impermeable epithelial barrier may contribute to the smaller water content of airway surface liquid (ASL) in cystic fibrosis. In this study, the feasibility and effectiveness of delivering an inhibitor of Na+ absorption, amiloride, by aerosol to the ASL of awake sheep was evaluated. Aerosolization of 6 X 10(-3) M amiloride, with [14C]amiloride, resulted in an estimated concentration of 7 X 10(-4) M in ASL. The transtracheal electric potential difference (PD), an index of the rate of Na+ absorption, was significantly inhibited (-29%) immediately after aerosolization of amiloride. The inhibition was short-lived and was correlated with rapid disappearance of the drug from ASL (half-time, 10.5 min between 0 and 30 min after aerosolization). Amiloride but not aerosol vehicle increased ASL volume significantly. The unidirectional flow of [14C]amiloride from the lumen to the submucosa across excised, short-circuited sheep airways exceeded flow from submucosa to lumen by a factor of at least 6. This finding suggests that amiloride is actively absorbed and is consistent with the rapid clearance from the airway surface and the short-lived effects of aerosolized amiloride in vivo. We conclude that sufficient concentrations of amiloride can be delivered to sheep airways by aerosol to inhibit PD and Na+ absorption for short periods.

Aerosols↗
PubMed1986

Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation.

The transepithelial potential difference (PD) of cystic fibrosis (CF) airway epithelium is abnormally raised and the Cl- permeability is low. We studied the contribution of active Na+ absorption to the PD and attempted to increase the Cl- permeability of CF epithelia. Nasal epithelia from CF and control subjects were mounted in Ussing chambers and were short-circuited. The basal rate of Na+ absorption was raised in CF polyps compared with control tissues. Whereas beta agonists induced Cl- secretion in normal and atopic epithelia, beta agonists further increased the rate of Na+ absorption in CF epithelia without inducing Cl- secretion. This unusual effect is not due to an abnormal CF beta receptor because similar effects were induced by forskolin, and because cAMP production was similar in normal and CF epithelia. We conclude that CF airway epithelia absorb Na+ at an accelerated rate. The abnormal response to beta agonists may reflect a primary abnormality in a cAMP-modulated path, or a normal cAMP-modulated process in a Cl- impermeable epithelial cell.

Adenylyl Cyclases↗
PubMed1986

Oxygen consumption and ouabain binding sites in cystic fibrosis nasal epithelium.

Ion transport by the epithelium lining the airways of patients with cystic fibrosis (CF) is characterized by a raised transepithelial PD and an increased amiloride sensitivity (1). These properties could arise from normal sodium transport across an epithelium with decreased cell chloride permeability and limited chloride secretion. Alternatively, a higher than normal rate of sodium absorption could contribute to these abnormalities. We investigated the latter possibility by measuring oxygen consumption and specific ouabain binding of CF and atopic polyp epithelia and normal turbinate epithelium. Tissue from CF patients consumed oxygen at a rate that was two to three times that of non-CF tissues and had 60% more ouabain binding sites than non-CF epithelium. These results are not consistent with an isolated defect in chloride permeability but support recent findings that the sodium conductance of the apical cell membrane and net sodium absorption by CF nasal epithelium are greater than those of non-CF nasal epithelium.

Adolescent↗