Porcine Trachea Research Articles

Involvement of Ca2+ mobilization in tachyphylaxis to beta-adrenergic receptors in trachealis.

We examined the mechanisms underlying tachyphylaxis to beta-adrenergic receptor agonists (beta-agonists) in tracheal smooth muscle. Simultaneous measurements of isometric tension and intracellular Ca2+ concentration ([Ca2+]i) using fura-2-loaded guinea pig tracheas showed that the inhibitory effects of isoproterenol (ISO) on tension and increases in [Ca2+]i induced by methacholine exhibited marked tachyphylaxis with repeated exposure to ISO at intervals of 15 min. Similarly, the activation of single Ca2+-activated K+ (KCa) channels in on-cell patches by 1 microM ISO was gradually attenuated after repeated extracellular application of ISO to single smooth cells of porcine tracheas. Desensitization of beta-adrenergic receptor/KCa channel stimulatory coupling and relaxation responses was prevented by separately antagonizing the voltage-dependent Ca2+ channel (VDCC) with verapamil, suggesting a surprising relationship between Ca2+ influx through VDCC and beta-adrenergic desensitization. Conversely, repeated exposure of 10 U/ml protein kinase A to inside-out patches did not result in desensitization of channel activation, and repeated exposure to 10 microM forskolin modestly augmented the inhibitory effects of forskolin on tension and [Ca2+]i by methacholine, indicating that the mechanism of desensitization is mediated by the beta-adrenergic receptor/G protein complex. These results indicate that an uncoupling of beta-adrenergic receptor from KCa channels augments Ca2+ mobilization through VDCC and stimulates tachyphylaxis.

Liquid secretion inhibitors reduce mucociliary transport in glandular airways

Because of its possible importance in cystic fibrosis (CF) pulmonary pathogenesis, the effect of anion and liquid secretion inhibitors on airway mucociliary transport was examined. When excised porcine tracheas were treated with ACh to induce gland liquid secretion, the rate of mucociliary transport was increased nearly threefold from 2.5 +/- 0.5 to 6.8 +/- 0.8 mm/min. Pretreatment with both bumetanide and dimethylamiloride (DMA), to respectively inhibit Cl(-) and HCO secretion, significantly reduced mucociliary transport in the presence of ACh by 92%. Pretreatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid similarly reduced mucociliary transport in ACh-treated airways by 97%. These agents did not, however, reduce ciliary beat frequency. Luminal application of benzamil to block liquid absorption significantly attenuated the inhibitory effects of bumetanide and DMA on mucociliary transport. We conclude that anion and liquid secretion is essential for normal mucociliary transport in glandular airways. Because the CF transmembrane conductance regulator protein likely mediates Cl(-), HCO, and liquid secretion in normal glands, we speculate that impairment of gland liquid secretion significantly contributes to defective mucociliary transport in CF.

Tracheal reconstruction with a synthetic material in a porcine model.

The technique of tracheal reconstruction with costal cartilage or a pericardial patch has resulted in successful management of many patients with tracheal stenosis. Yet, it is often difficult, if not impossible, to harvest enough cartilage in a neonate. Pericardial patches are flimsy and can collapse. This pilot study evaluated the use of absorbable plates made of an alloplastic material, Lactosorb, as a substitute for cartilage on a growing porcine trachea. The rectangular plates were fashioned into an inverted U and placed into the airway through an anterior tracheal split. This placement resulted in granulation formation and failure. The plates were then fashioned into an I configuration and placed inside and outside the tracheal lumen. This attempt also resulted in failure, secondary to respiratory distress. Finally, the plates were fashioned into an inverted U and placed outside the trachea. In 67% of the subjects, the distraction was maintained and growth continued for the duration of the study. The stent was completely absorbed, with minimal scar or granulation tissue, in 4 months. In 1 subject, the stent migrated, and 1 subject died of a mucous plug. This study suggests that Lactosorb has no role in intraluminal stenting, but that it may be a viable alternative to a pericardial patch or cartilage in tracheal reconstruction when it is placed outside the airway lumen in a young animal.

Localization of porcine trappin-2 (SKALP/elafin) in trachea and large intestine by in situ hybridization and immunohistochemistry.

Trappin-2 (SKALP/elafin), an elastase inhibitor, belongs to a unique family of proteinase inhibitors that are covalently anchored at the site of action through their transglutaminase substrate domain and are collectively called trappins. The transglutaminase substrate domain is therefore called "cementoin moiety". Currently, human, porcine, and bovine trappin-2 (SKALP/elafin) have been characterized. Previously, we showed that porcine trappin-2 (SKALP/elafin) occurs mainly in the trachea and large intestine. To determine the localization of trappin-2 (SKALP/elafin) at the cellular level, we carried out in situ hybridization and immunohistochemistry using the porcine trachea and large intestine and found that trappin-2 (SKALP/elafin) is produced in the goblet cells of the tracheal epithelium and of the large intestinal crypts. These locations suggest that trappin-2 (SKALP/elafin) is secreted onto the luminal surface of the trachea and crypts of Lieberkuhn and plays a protective role against destructive bacterial proteinases.

1H and 13C nuclear magnetic resonance identification and characterization of components of chondroitin sulfates of various origin

Three natural chondroitin sulfates (CSs), from porcine and bovine trachea, and from shark cartilage, were studied using a variety of NMR techniques (DQS, TOCSY, NOESY, HMQC). A good 1H and 13C characterization of the major components, chondroitin 4-sulfate (CS4) and chondroitin 6-sulfate (CS6), was obtained and a number of signals coming from chondroitin 2,6-disulfate (CS2,6) (present only in shark CS) was identified. The study of a chemically desulfated CS was necessary in order to understand the difficulties encountered in detecting signals from the chondroitin non-sulfate (CS0) component of porcine and bovine CSs. The singular pattern of UC-4 and NC-1 signals was recognized and explained in terms of a diad model. The “excess of multiplicity” affecting mainly these two signals was attributed to the differences in the conformation of the N 1:4 U glycosidic bond. Further support to this hypothesis comes from the comparison of the NOESY spectra of the three CSs.

Expression and function of endothelins, endothelin receptors, and endothelin converting enzyme in the porcine trachea.

Endothelins (ETs) can modulate the airway smooth muscle tone. Using simultaneous measurements of cytosolic Ca2+ concentration ([Ca2+]i) and tension as well as the reverse transcription polymerase chain reaction (RT-PCR), we examined ET systems in the porcine trachea. In the functional study, the application of ET-1, ET-3 or sarafotoxin S6c (S6c) caused increases in [Ca2+]i and tension, in a concentration-dependent manner. These ET ligands were found to increase the Ca2+ sensitivity of the myofilament of the tracheal smooth muscle cells (SMCs). The contractions induced by ET-1 (10(-7) M), an ET receptor (ET-R) non-selective agonist, were much greater than those induced by S6c, an ET(B)-R selective agonist. BQ-123 (10(-6) M), an ET(A)-R antagonist, inhibited the ET-1 induced contraction. These functional experiments suggested the presence of both functioning ET(A)- and ET(B)-Rs in tracheal SMCs. RT-PCR experiments revealed that the tracheal SMCs expressed both ET(A)-R and ET(B)-R mRNAs, while tracheal epithelial cells (EpCs) predominantly expressed ET(A)-R mRNA. The porcine tracheal SMCs and EpCs also expressed pre-pro ET-1 (ppET-1), ppET-3, and endothelin converting enzyme-1 (ECE-1) mRNAs. These results suggested that ETs induce contraction of porcine tracheal SMCs not only by increasing [Ca2+]i but also increasing the Ca2+ sensitivity of the myofilament and that ETs could potentially be the autocrine and/or paracrine transmitters to regulate the contraction in the porcine airway smooth muscle.

Caffeine- and Inositol 1,4,5-Trisphosphate-Induced45Ca2+Releases in the Microsomes of Tracheal Epithelial Cells

Major parts of microsomes prepared from the epithelial cells of porcine trachea were tight-sealed vesicles since they showed a saturation of45Ca2+uptake and spontaneous releases of stored45Ca2+by the treatments of Ca2+-ionophore and Ca2+channel agonists. In the presence of caffeine (10 mM), the maximal release of microsomal45Ca2+was observed at the extramicrosomal Ca2+concentrations between 0.1∼1 μM and at below or above this range of Ca2+concentration the releases were decreased, forming a bell-shaped curve. These results indicate that the microsomal45Ca2+releases were mediated by ryanodine receptor, a caffeine-sensitive Ca2+channel. Caffeine (10 mM) released 30.2±5.9% of microsomal45Ca2+while inositol 1,4,5-trisphosphate (InsP3, 10 μM) released 18.4±3.0% of the stored45Ca2+. Caffeine-induced and InsP3-induced45Ca2+releases were additive, implying that these two types of45Ca2+releases are from physically distinct microsomes. Procaine, an antagonist of ryanodine receptor, selectively blocked the effect of caffeine but not the effect of InsP3. The results suggest that the epithelial cells of porcine trachea have caffeine-sensitive Ca2+store in addition to InsP3-sensitive one.

Regional bioelectric properties of porcine airway epithelium

Ion transport properties of pulmonary small airway epithelia are poorly understood. To characterize these properties, airways were excised from anesthetized pigs. Transepithelial potential difference (PD) and conductance were measured in five airway regions: trachea (T, 7.9 +/- 0.2 mm diam), mainstem bronchi (MB, 5.5 +/- 0.2 mm diam), large bronchi (LB, 1.69 +/- 0.12 mm diam), small bronchi (SB, 0.70 +/- 0.06 mm diam), and bronchioles (BR, 0.25 +/- 0.05 mm diam). T and MB were mounted in Ussing-type chambers, and LB, SB, and BR were cannulated with pipettes and perfused. PDs of control tissues were -9.7 +/- 0.8 mV (T), -4.0 +/- 0.5 mV (MB), -4.3 +/- 1.0 mV (LB), -4.5 +/- 0.4 mV (SB), and -1.5 +/- 0.4 mV (BR), lumen negative. Amiloride significantly (P < 0.05) inhibited PDs by 25-70% in all airway regions and decreased conductance 17-33% in all regions except LB where a 10% increase was observed. Bumetanide significantly reduced the amiloride-insensitive PD 54-62% in all regions except BR. Bumetanide had little effect on conductance in T, SB, and BR, but conductance was increased in MB and LB. All airways except the smallest BR significantly hyperpolarized when the solution that bathed the lumen was replaced with Cl(-)-free solution. In bronchioles, hyperpolarization by luminal Cl(-)-free solution was inversely related to fractional inhibition of PD with amiloride but directly related to lumen diameter. These results suggest that 1) porcine tracheas, bronchi, and bronchioles actively absorb Na+, and 2) secretion of Cl- may occur in all airway regions except small bronchioles.

Mechanism of relaxing action of the antiasthmatic drug, azelastine, in isolated porcine tracheal smooth muscle

Azelastine (1–300 μM) inhibited contractions of isolated porcine trachea induced by high K +, carbachol and endothelin-1 (ET-1) with a decrease in [Ca 2+] cyt (as measured by fura-2-fluorescencc). Verapamil (0.1–10 μM) also inhibited the high K +-induced increases in [Ca 2+] cyt and contraction, although it only partially inhibited the responses evoked by carbachol or ET-1. In the absence of extracellular Ca 2+ (with 0.5 mM EGTA). carbachol induced a transient increase in [Ca 2+] cyt, and force by releasing Ca 2+ from cellular stores. Azelastine (100 μm) completely inhibited these contransient changes. In the absence of extracellular Ca 2+, carbachol and 12-deoxyphorbol 13-isobutyrate (DPB) induced small sustained contractions without increasing [Ca 2+] cyt. Azelastine inhibited these contractions. In muscle permeabilized with α-toxin, Ca 2+ (0.3–3 μM) induced contraction in a concentration-dependent manner. DPB (without GTP) and carbachol or ET-1 (with GTP) enhanced the Ca 2+-induced contraction. Azelastine partially inhibited the contraction induced by 0.3 μM Ca 2+ but not the contraction induced by 3 μM Ca 2+, and strongly inhibited the potentiating effects of DPB, carbachol and ET-1. Azelastine had no effect on the content of cyclic AMP or cyclic GMP. These results suggest that azelastine inhibits smooth muscle contraction by (i) decreasing [Ca 2+] cyt, by inhibition of Ca 2+ channels, (ii) decreasing agonist-induced Ca 2+ release, and (iii) direct inhibition of contractile elements.

Ozone absorption into excised porcine and sheep tracheae by a bolus-response method

The absorption of ozone (O3) into excised porcine and sheep tracheae was characterized by a bolus-response experiment in which a bolus with a peak O3 concentration of 1 ppm was rapidly injected into a steadily flowing airstream entering the trachea. Using a fast-responding chemiluminescent analyzer of our design, the O3 concentration curves at the proximal end (i.e., the bolus input) and at the distal end (i.e., the response) of the trachea were monitored. Each concentration curve was numerically integrated, and the fraction of O3 absorbed in the trachea was obtained by subtracting from unity the ratio of the response integral to the bolus input integral. Average values of ozone-absorbed fraction decreased from about 0.50 to 0.15 at increasing airflows from 50 to 200 ml/sec. A diffusion theory that includes the effects of bulk convection, axial dispersion, and first-order absorption was developed to relate the fractional absorption to an overall mass transfer coefficient (K). The results indicate that K is independent of airflow, suggesting that the diffusion resistance in mucus is much greater than that in the gas phase. The time-weighted integrals of the concentration curves were also computed, allowing the mean residence time of O3 in the trachea (delta tau) to be determined. As predicted by the diffusion theory, delta tau was inversely related to the rate of O3 absorption.

Residual strains in porcine and canine trachea

Residual strains exist in canine and porcine tracheas. They are revealed by cutting the trachea first perpendicular to its axis into rings, then radially into sectors. Each sector is characterized by an opening angle which is defined as the angle subtended between two radii joining the middle point of the inner wall to the tips of the inner wall. The trachea being non-axisymmetric, the opening angle depends on the position of the radial cut. The trachea being also nonuniform in the axial direction, the opening angle varies along the length of the trachea. In the dog, the opening angle of the trachea cut at the anterior position (cartilaginous) is about 100° at the larynx; it increases fairly linearly to 180° midway down the trachea; then increases slowly to about 200° at the lower end where the trachea bifurcates into the main bronchi. Dog trachea cut in the posterior (muscular) position have an opening angle of about 50° at the larynx, which increases to about 70° three-quarters of the way down the trachea, then drops to 60° at the lower end. In the pig, the opening angle of the trachea is much smaller, the values at anterior and posterior cuts are similar (without significant difference), and their mean value decreases from about 15° at the laryngeal end to about 5° at the lower end. These species and regional differences are discussed in relation to tracheal geometry and structure. It is shown that one radial cut reduces a tracheal ring of either dog or pig into zero-stress state; and that the opening angle obtained by cutting the ring at one point can be computed from that obtained by cutting at another point if the geometry of the tracheal sector is known at a zero-stress state.

Smooth Muscle Mechanics: Implications for Airway Hyperresponsiveness

Human bronchial smooth muscle preparations from 10 freshly dissected pneumonectomy samples were evaluated for their mechanical characteristics and compared with results obtained for similar samples obtained from porcine trachea. Length-tension relationships of in vitro smooth muscle were evaluated for passive stretching as well as active isometric force generation and isotonic shortening using electrical field stimulation. At the length (Lmax) producing maximal force (Pmax) resting tension was very high (60.0 +/- 8.8% Pmax) compared with porcine trachealis (5.2 +/- 2.3% Pmax). Maximum shortening was 25.0 +/- 9.0% at a length of 75% Lmax with suboptimal shortening occurring at Lmax (12.0 +/- 3.4%) for the human bronchus, whereas optimal shortening of porcine trachealis (71.4 +/- 3.6%) occurred at Lmax. Morphometric evaluation revealed threefold less muscle per cross-sectional area of tissue for human (8.7 +/- 1.5%) versus porcine (24.8 +/- 1.9%) preparations. We conclude that the high passive tension and the decreased maximum shortening are produced by a relatively large load which must be overcome for the muscle to shorten, presumably provided by the greater connective tissue elastic component present in the airway. We suggest that a decrease in airway wall elastance would increase smooth muscle shortening, thereby leading to excessive responses to contractile agonists as seen in vivo in asthma.

P-332 - Effects of vasoactive intestinal polypeptide (VIP) and isoproterenol (Iso) on membrane currents in single smooth muscle cells isolated from porcine trachea.

P-332 - Effects of vasoactive intestinal polypeptide (VIP) and isoproterenol (Iso) on membrane currents in single smooth muscle cells isolated from porcine trachea.

Dynamics of sulfur dioxide absorption in excised porcine tracheae

The absorption of sulfur dioxide (SO2) into excised porcine tracheae was characterized by a step-response experiment in which SO2 outlet concentration was monitored during the 30-min interval following introduction of inlet concentrations of 0.1-0.6 ppm at steady air flows of 2.7-11.0 liter/min. These data were analyzed with a diffusion-reaction theory incorporating three independent parameters--a gas phase mass transfer coefficient, kg, a tissue phase diffusivity x solubility product, D(alpha RT)2, and a tissue phase reaction constant, kr. While single values of 17 sec-1 for kr and 0.28 m2/sec for D(alpha RT)2 were sufficient to simulate all the data, it was necessary to vary kg from a 0.032 to 0.121 m/sec in direct proportion to the gas flow. Based on these parameter values, gas phase resistance accounts for about one-fourth of the total resistance to absorption in gas and tissue phases combined. All three parameters were independent of inlet concentration, implying that diffusion, solubility, and irreversible reaction of SO2 in tissue are all linear processes.

Adherence of Bordetella bronchiseptica 276 to porcine trachea maintained in organ culture

Two organ culture models have been adapted for porcine tracheae in order to study colonization by Bordetella bronchiseptica. Rings or segments excised from tracheae of newborn piglets were incubated overnight at 37 degrees C in a nutrient medium under 5% CO2-95% air conditions. Tracheal segments were infected with B bronchiseptica 276, and after different incubation times, bacterial counts were done. B. bronchiseptica adhered well to tracheae maintained in culture, and no statistically significant differences between the two models were observed. Noninfected tracheal mucosae maintained a normal appearance for several days, whereas infected mucosae showed typical damage caused by B. bronchiseptica, namely, loss of ciliary activity and cilia and sloughing of ciliated cells. Our data indicated that porcine tracheal organ culture could be advantageously used to study in vitro colonization by B. bronchiseptica.

Inhibition and recovery of mammalian respiratory ciliary function after formaldehyde exposure

Formaldehyde (HCHO) has been reported to impair mucociliary clearance. The present investigation using rabbit and porcine tracheal explants in vitro examined (1) the impairment of ciliary activity, an essential component of mucociliary clearance; (2) the reversibility of ciliary dysfunction after HCHO exposure; and (3) the mechanism by which ciliary activity is reduced by HCHO. HCHO treatment of rabbit tracheal rings significantly decreased zones of active ciliated epithelium in direct proportion to concentration and exposure duration. There was also a significant concentration-dependent reduction of ciliary beat frequency. Removal of HCHO permitted recovery of zones of ciliary activity to normal beat frequencies; greater inhibitory concentrations of HCHO required greater time for return of function. Treatment of porcine tracheae with increasing concentrations of HCHO for time periods inhibitory to rabbit ciliary activity correspondingly reduced the yield of cilia extractable from treated epithelium. Furthermore, the specific activity of ATPase of extracted ciliary axonemes was diminished with increasing HCHO concentration, indicating loss of function. A recovery period following identical exposures of the porcine tracheae to the lower HCHO concentrations resulted in normal yields of functionally intact ciliary axonemes. Similarly, a recovery period after the highest HCHO concentration produced more functional axonemes than obtained from exposed tracheae without a recovery period, although less than normal yields. Therefore, ciliary dysfunction elicited by a defined range of HCHO concentrations is reversible. The yield and functional integrity of ciliary axonemes from epithelium exposed to HCHO with a recovery period are significantly greater than those without such a recovery period, suggesting an alteration and subsequent repair of epithelial surface components following HCHO exposure.

Quantitative measurement of smooth muscle shortening in isolated pig trachea.

Matched porcine tracheal rings were exposed to theophylline and increasing doses of carbachol in Krebs solution. Histological sections of each ring were traced and each of the following dimensions measured: the external perimeter (Pe) and external area (Ae) defined by the outer border of smooth muscle and inner surface of cartilage, and the internal perimeter (Pi) and internal area (Ai) defined by the luminal surface of the epithelium and the muscle length (L) along its outer border. Absolute wall area (WA = Ae - Ai) and relative wall area (PW = WA/Ae) were calculated. Carbachol-treated tracheal ring dimensions were compared with those of their matched theophylline-treated rings. In tracheal rings with intact cartilage, maximal smooth muscle shortening of 44% was achieved with 10(-2) M carbachol. In tracheal rings in which anterior and posterior segments of cartilage were excised, the trachealis muscle passively shortened by 20% and maximal shortening (10(-3) M carbachol) was 57%. Although Ai decreased with maximal smooth muscle shortening, there were no changes in the length of Pi or in WA. These data show that the cartilage in the porcine trachea exerts both a preload that passively stretches the trachealis muscle and an afterload that limits maximal smooth muscle shortening.

Platelet activating factor (PAF) is a potent stimulator of porcine tracheal fluid secretion in vitro

Mocus hypersecretion is a major clinical feature of chronic obstructive lung diseases such as asthma. The possible role of the inflammatory and bronchoconstrictor ether lipid PAF (platelet activating factor) has been studied in isolated porcine trachea with the tantalum ‘hillock’ technique used to visualize fluid production from tracheal submucosal glands. PAF caused a rapid, dose-dependent (0.001-1 nM) stimulation of fluid secretion which could be detected after 5 min and which increased with time up to at least 15 min. The PAF-induced fluid secretion was unaffected by both antagonists of histamine, acetylcholine and leukotriene D 4 and inhibitors of prostaglandin and leukotrienes synthesis. A purported PAF receptor antagonist (CV 3988) inhibited the PAF responses in a dose-dependent manner implying a receptor-mediated event. These results may be of relevance to the mucus hypersecretion seen in chronic airway diseases.

Disruption of respiratory cilia by proteases including those of Pseudomonas aeruginosa.

Pseudomonad proteases disrupted the function and structure of demembranated cilia (axonemes) extracted from porcine tracheae. Proteolytic degradation by the two pseudomonad proteases elastase and alkaline protease and by trypsin and subtilisin impaired motility of ATP-activated axonemes. In addition, electron microscopic observation of negatively stained axonemes indicated that exposure to proteases caused dissociation into individual doublet or singlet microtubules. Inhibition of motility and axonemal fraying occurred when axonemes were treated with less than 5 U of proteolytic activity of any of the four proteases tested. When the effects of 2 U of each protease were compared, trypsin and subtilisin were able to produce immotility in less time than pseudomonad elastase and alkaline protease, while alkaline protease and subtilisin caused the most axonemal fraying in 10 min. Proteolytic digestion of axonemal proteins was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All four proteases cleaved dynein proteins (proteins necessary for motility), though treatment with trypsin resulted in the most extensive solubilization of axonemal proteins. Trypsin and subtilisin both produced more changes in the protein profiles of treated axonemes, using fewer units of proteolytic activity, than the pseudomonad proteases. However, the limited alteration of only a few axonemal proteins by pseudomonad proteases indicates that cleavage need not be extensive to produce dysfunction. Thus, ciliary axonemes are susceptible to proteolytic attack. Degradation of axonemal proteins by pseudomonad proteases, which are released during active infection, may contribute to the impaired ciliary function associated with pseudomonad colonization of the respiratory tract.

Isolation of cilia from porcine tracheal epithelium and extraction of dynein arms.

Milligram amounts of mammalian ciliary axonemes were isolated from porcine tracheas. These were reactivated upon addition of ATP, indicating intact functional capability with a mean beat frequency at 37 degrees C of 8.2 Hz. Electron microscopy showed typical ultrastructure of the isolated demembranated axonemes. Electrophoresis into polyacrylamide gradient gels containing sodium dodecyl sulfate revealed reproducible protein profiles from ten different tracheal preparations. Four major protein bands were observed in the 300-330 K molecular weight region, as well as tubulin at 51-54K. Extraction of the isolated tracheal axonemes with 0.6M KCl removed the outer dynein arms seen in electron microscopic cross-section of axonemes, preferentially solubilized two of the high molecular weight proteins at 320 and 330 K, and resulted in a three- to four-fold increase in ATPase specific activity. Sedimentation of the dialyzed salt extract on a 5-30% sucrose density gradient and subsequent fractionation yielded two peaks of ATPase activity. The faster migrating, 19S major ATPase peak correlated with the 320 and 330 K proteins, and two other proteins at 81 and 67 K. The slower sedimenting, 12S minor ATPase peak corresponded to a 308 K protein and two smaller proteins at 33 and 48 K. Thus, the outer dynein arm of tracheal cilia appeared to be associated with at least two high molecular weight proteins. These results demonstrate that adequate quantities of functionally intact axonemes can be reproducibly isolated from porcine tracheas, allowing further fractionation and analysis of mammalian cilia.