Donovan B. Yeates

Bidirectional Transepithelial Water Transport: Measurement and Governing Mechanisms

In the search for the mechanisms whereby water is transported across biological membranes, we hypothesized that in the airways, the hydration of the periciliary fluid layer is regulated by luminal-to-basolateral water transport coupled to active transepithelial sodium transport. The luminal-to-basolateral (JWL-->B) and the basolateral-to-luminal (JWB-->L) transepithelial water fluxes across ovine tracheal epithelia were measured simultaneously. The JWL-->B (6.1 microliter/min/cm2) was larger than JWB-->L (4.5 microliter/min/cm2, p < 0.05, n = 30). The corresponding water diffusional permeabilities were PdL-->B = 1.0 x 10(-4) cm/s and PdB-->L = 7.5 x 10(-5) cm/s. The activation energy (Ea) of JWL-->B (11.6 kcal/mol) was larger than the Ea of JWB-->L (6.5 kcal/mol, p < 0.05, n = 5). Acetylstrophanthidin (100 microM basolateral) reduced JWL-->B from 6.1 to 4.4 microliter/min/cm2 (p < 0. 05, n = 5) and abolished the PD. Amiloride (10 microM luminal) reduced JWL-->B from 5.7 to 3.7 microliter/min/cm2 (p < 0.05, n = 5) and reduced PD by 44%. Neither of these agents significantly changed JWB-->L. These data indicate that in tracheal epithelia under homeostatic conditions, JWB-->L was dominated by diffusion (Ea = 4.6 kcal/mol), whereas approximately 30% of JWL-->B was coupled to the active Na+,K+-ATPase pump (Ea = 27 kcal/mol).

Generation of concentrated aerosols for inhalation studies

Abstract A technique is described for generation of concentrated respirable aerosols and their administration to canine lungs. The functional characteristics of the system were delineated using aqueous solutions of disodium fluorescein (DF). The aerosol was generated by delivering pressurized air and disodium fluorescein solution to a Turbotac jet nebulizer. The particles were dried with a sheath of warm air and concentrated using a seven orifice virtual impactor. The input aerosol particles were concentrated up to eight times, resulting in output aerosol concentration of about 10 mg m−3 at a flow rate of 20 l min−1. The particles had predictable mass median aerodynamic diameters between 4 and 7 μm, and geometric standard deviations between 1.7 and 2.0. To conduct inhalation studies on beagle dogs, the aerosol generation and concentration system was pressurized to 18 cm of water. In these experiments the test aerosol consisted of a mixture of disodium fluorescein with technetium-99m tagged to iron oxide colloid, controlled delivery of the aerosol to the dogs was achieved by sequencing low resistance solenoid valves using a logic control box. The animals were anesthetized and endotracheally intubated. The aerosols were carried in the ventilating air stream. The duration of exposure was 3 min. Gamma scintigraphy confirmed deposition of the aerosol in the lower lungs as demonstrated by a total lung retention between 70 and 75% after 24 h. This methodology is applicable for use with solutions and/or colloidal suspensions and can be adapted for continuous aerosol generation and delivery.

Controlled-Release from Condensation Coated Respirable Aerosol-Particles:

Extending the residence time of drugs delivered to the lungs as inhalation aerosols may result in sustained therapeutic drug levels and reduced toxicity. Droplets were generated from 0.25 wt% disodium fluorescein (DF), and 0.25 wt% albuterol sulphate solutions at a rate of 1 ml min−1 using a Turbotac jet nebulizer. These droplets were dried, concentrated and mixed with saturated lauric acid (LA) vapor at bath temperatures of 60–140°C. The resulting coated particles were < 5 μm in size as estimated by inertial impaction and scanning electron microscopy. Powder composition, as determined by gas chromatography, ranged from ratios of 1.2:1 to 2.5:1, of LA: DF. Evidence of coating of DF by LA was derived from i.r. spectroscopy and X-ray microanalysis. Dissolution studies performed on the coated particles in phosphate buffer, pH 7.4 at 37°C and quantified by u.v. spectroscopy, showed that the half-time for dissolution (t2) increased from 4 ± 2 min for uncoated DF particles, to 22 ± 3 − 55 ± 2 min for lauric acid coated DF particles, depending on the coating thickness. The t12 for albuterol sulphate particles increased from 2.5 ± 1.5 min to 12.5 ± 1.9 min for albuterol sulphate particles coated with lauric acid at a bath temperature of 100°C. Inhalation studies performed on beagle dogs with DF particles coated with lauric acid (bath temperature, 100°C) indicated there was a shift and broadening of the peak plasma concentration in comparison with aerosols of DF alone. The average absorption half-time increased from 4.7 ± 0.8 min for uncoated DF particles to 11.5 ± 1.6 min for lauric acid coated DF particles.

Stationary and nonstationary correlation-frequency analysis of heterodyne mode laser light scattering: magnitude and periodicity of canine tracheal ciliary beat frequency in vivo

Stationary and nonstationary correlation-frequency analysis of heterodyne laser light scattering were utilized to make automated, on-line, objective measurements of tracheal ciliary beat frequency (CBF) in intact, anesthetized canines. The stationary correlation-frequency analysis laser light-scattering technique was used to assess the magnitude of the CBF stimulatory responses induced by aerosolized 10(-5) M fenoterol (sympathomimetic), and 10(-8) M and 10(-6) M methacholine (parasympathomimetic) delivered to the whole lungs of eight barbiturate-anesthetized beagles. The nonstationary correlation-frequency analysis laser light-scattering technique was used to measure the effect on tracheal CBF of increasing the cytosolic calcium ion concentration with a calcium ionophore, A23187. Aerosolized A23187 was delivered to the isolated tracheal lumens of eight beagle dogs in cumulative doses ranging from 10(-9)M to 10(-6) M. Administration of the ionophore synchronized the CBF with a period of 5.3 min. Dose dependencies were observed in both the time to the peak CBF stimulation and the magnitude of the stimulatory response. The magnitude of CBF stimulation was inhibited by prior administration of aerosolized nifedipine (2 mg/ml), a voltage-operated calcium channel blocker. The A23187-induced modulation period of tracheal CBF, was unchanged by nifedipine. These are the first data to demonstrate that the magnitude and periodicity of CBF are two independent coupled processes. The cooperativity of these two processes could be determined in the effectiveness of mucociliary transport.

Regulatory pathways for the stimulation of canine tracheal ciliary beat frequency by bradykinin.

1. The effects of bradykinin, a potent inflammatory nanopeptide, on tracheal ciliary beat frequency in vivo were investigated using barbiturate‐anaesthetized beagles. Tracheal ciliary beat frequency was measured using heterodyne mode correlation analysis laser light scattering, a technique that does not require surgical intervention. 2. Aerosolized 10(‐5) M‐bradykinin in 0.9% saline administered for 3 min to eight barbiturate‐anaesthetized beagles stimulated tracheal ciliary beat frequency from the baseline of 5.3 +/‐ 0.1 Hz to a maximum of 16.6 +/‐ 2.0 Hz, 8 min after aerosol delivery, and ciliary beat frequency remained above baseline for the following 35 min. 3. Intravenously injected hexamethonium bromide, ipratropium bromide or indomethacin did not change baseline tracheal ciliary beat frequency. That down‐regulation of ciliary beat frequency below baseline values was not observed with either the neural or the cyclooxygenase blocking agents suggests that neither of these pathways is involved in the maintenance of the observed basal ciliary beat frequency. 4. Bradykinin‐induced stimulation of tracheal ciliary beat frequency is blocked by hexamethonium bromide, ipratropium bromide or indomethacin. These data suggest that the stimulation of ciliary beat frequency by bradykinin acts through both cellular cyclooxygenase and parasympathetic pathways in series.

Rotating magnetic particle microrheometry in biopolymer fluid dynamics: mucus microrheology.

The polymer properties of canine mucus were investigated through the method of rotating magnetic particle microrheometry. Mucus is visualized as a physically entangled biopolymer of low polydispersity in a water-based solution. Mucus was modeled according to the constitutive law of a Doi-Edwards fluid. The magnetic-particle equation of rotational motion is analytically solved in the linear viscoelastic limit rendering theoretical flow profiles which are used to fit the experimental trace signals of the particle remanent-magnetic-field decay. The zero-shear-rate viscosity was found to be 18,000 P and the relaxation time at about 42 s. The molecular weight between entanglements for mucins was estimated at 1.7 MDa rendering an estimation of about seven physical cross-links per molecule. Rheological investigations were extended also to diluted and concentrated rations of the normal mucus simulating the conditions found in more physiological extremes.

Tracheal ciliary beat frequency in baboons : effects of peripheral histamine and capsaicin

To determine if tracheal ciliary beat frequency (CBFt) in the nonhuman primate could be stimulated indirectly, either by an inhaled irritant or by a mediator potentially released from an inhalation challenge, saline (0.9%), capsaicin (3×10−9M), or histamine (8 mg/ml) were delivered for three minites as 100 ml aerosol (0.25 μm MMAD) boluses at the beginning of each breath, followed by 10 sec breath hold. Each of five adult baboons (17–27 kg,Papio cynocephalus) underwent three studies using a randomized block design. The baboons were anesthetized and ventilated eucapnically through an endotracheal tube whose cuff was inflated in the distal trachea. CBFt was measured in the mid-trachea proximal to the cuff at one minute intervals using heterodyne laser light scattering. There was no change in CBFt from a baseline of 7.2±0.6 Hz (mean ± SE) following inhalation of saline deposited peripherally. Capsaicin stimulated CBFt for over 80 min reaching a maximum of 14.7±6.3 Hz 46 min after aerosol delivery. Histamine stimulated CBFt for 90 minutes reaching a maximum of 19.3±9.9 Hz 67 min after aerosol delivery. The prolonged stimulatory responses of CBFt to peripherally deposited capsaicin and histamine most likely involve both cellular and neural pathways through which the tracheobronchial airways, in response to an alveolar challenge, are cleansed of secretions thus enabling the maintenance of effective gas transport.

Rotational magnetic particle microrheometry: The Newtonian case

A rotating sphere microrheometer, based on extensions of the work of Valberg [Valberg, P.A., “Magnetometry of Ingested Particles in Pulmonary Macro-Phages,” Science 224, 513–516 (1984); Valberg, P.A., and H. A. Feldman, “Magnetic Particle Motions within Living Cells: Measurement of Cytoplasmic Viscosity and Motile Activity,” Biophys. J. 52, 551–572 (1987)] and Edwards and Yeates [Edwards, P.A., and D. B. Yeates, Viscoelasticity of Biomaterials, Chap. 16, ACS Symposium Series, 489, edited by W. Glasser and H. Hatakeyama (Boston, MA, 1992)], was developed to rapidly (within 10 s) measure the rheological properties of small (∼10 μL) quantities of highly viscous (100–10 000 poise) fluids at small (10−3–10−1 Hz) rates of strain. Previous experimental work was extended by the use of MQP-B™ 1.4-μm-radius particles, which have extremely high coercivity and remanent magnetic field, and in which rotation of magnetic domains within the particle does not occur. The microrheometer was tested with a series of Newtonian viscosity standards (100–10 000 poise) and found to accurately predict viscosity (error range 3%–9%). The effects of shape, size distribution, sedimentation, particle–particle magnetic interactions, and agglomeration were investigated and found to be either negligible or easily determined. This microrheometer can be used to determine the rheological properties of minute quantities of viscous fluids and may be applicable to the measurement of zero-shear-rate viscosity of viscoelastic fluids.A rotating sphere microrheometer, based on extensions of the work of Valberg [Valberg, P.A., “Magnetometry of Ingested Particles in Pulmonary Macro-Phages,” Science 224, 513–516 (1984); Valberg, P.A., and H. A. Feldman, “Magnetic Particle Motions within Living Cells: Measurement of Cytoplasmic Viscosity and Motile Activity,” Biophys. J. 52, 551–572 (1987)] and Edwards and Yeates [Edwards, P.A., and D. B. Yeates, Viscoelasticity of Biomaterials, Chap. 16, ACS Symposium Series, 489, edited by W. Glasser and H. Hatakeyama (Boston, MA, 1992)], was developed to rapidly (within 10 s) measure the rheological properties of small (∼10 μL) quantities of highly viscous (100–10 000 poise) fluids at small (10−3–10−1 Hz) rates of strain. Previous experimental work was extended by the use of MQP-B™ 1.4-μm-radius particles, which have extremely high coercivity and remanent magnetic field, and in which rotation of magnetic domains within the particle does not occur. The microrheometer was tested with a series of Newtonian...

Alveolar epithelial permeability in baboons: histamine and capsaicin.

1. We investigated if changes in alveolar epithelial permeability could be initiated by various doses of the irritant capsaicin through stimulation of pulmonary afferent nerves either directly, or indirectly through the release of an intermediate cell mediator, and whether mediation of permeability involved histamine H1 and/or H2 receptors. 2. Alveolar epithelial permeability was indicated by the clearance of peripherally deposited 99mTc‐DTPA (technetium‐99m‐diethylenetriamine pentaacetate) aerosol in baboons. Aerosol challenge experiments were performed twice with histamine (32 mg/ml), once each with capsaicin (10(‐6), 10(‐5), 10(‐4) and 10(‐3) M), and once each with histamine (32 mg/ml) preceded by H1 (terfenadine) and H2 (ranitidine) antagonists alone and combined. 3. Mean half‐time for 99mTc‐DTPA clearance was 66 +/‐ 4 min. After histamine, it decreased to 41 +/‐ 5 min (P < 0.05), and after capsaicin it was 70 +/‐ 5 min (combined doses). Pretreatment with the H1 and H2 antagonists, either separately or in combination, did not consistently inhibit increases in 99mTc‐DTPA permeability. 4. Capsaicin caused tachypnoea only at 10(‐3) M (P < 0.05). Inhibition of histamine‐induced tachypnoea required both antagonists (P < 0.05) suggesting the existence of a population of peripheral sensory neurons possessing H1 and H2 receptors. Changes in permeability were unrelated to the changes in respiratory frequency. Thus, neither the mechanisms that stimulate respiratory frequency, nor the respiratory frequency per se, were responsible for increased alveolar epithelial permeability. 5. Capsaicin did not change epithelial permeability at doses that cause capsaicin‐sensitive C fibre stimulation as delineated by increases in respiratory frequency. Nor does C fibre stimulation, induced by mediators released from pulmonary cells activated by low doses of capsaicin, increase epithelial permeability. These data suggest that in the primate, histamine increases epithelial permeability to small solutes by a mechanism independent of the activation of capsaicin‐sensitive C fibres and capsaicin‐initiated release of cell mediators. 6. These findings do not support a role for C fibre activation in regulating epithelial permeability of small solutes.

A technique for measuring bronchial mucociliary clearance in unsedated dogs

Abstract An unsedated dog model of bronchial mucociliary clearance was developed using radioaerosol techniques. A neutralized, concentrated, monodisperse iron oxide aerosol (5–6 μm MMAD) radiotagged with technetium-99m was delivered to the dog via a face mask. Retained activity was measured while the dog was restrained in front of a gamma camera. Measurements were made for 2–3 h post-inhalation and again at 24 h. Three beagle dogs, each studied four times, demonstrated mean percents for 2 h particle retention within the whole lung of 76 ± 13, 92 ± 7 and 89 ± 4, within the bronchial airways of 48 ± 27, 84 ± 15 and 73 ± 10, and mean 24 h particle retentions of 47 ± 18, 65 ± 11 and 57 ± 9, respectively. The unsedated beagle dog provides a potential model for study of the effects of drugs, disease and inhaled pollutants on mucociliary transport.