On the mechanics of mucociliary flows. III. Flow-velocity profiles in frog palate mucus

Flow-velocity profiles over excised frog ciliated epithelium were obtained for the region within about 600 micron of the mucosa. Fluorescent particles were used as flow tracers. Both a control and an autologous mucus suspension were observed. The control culture medium was bounded by the walls of the observation chamber, and mucus was deposited on the epithelium as a blob after mixing it with tracers. In spite of the difference in boundary conditions the two profiles, normalized to maximum particle velocity and solution depth, were indistinguishable at heights over 60 micron from the mucosa. The near-mucosa profiles in contrast were unalike with mucus exhibiting a greater shear gradient than the control culture medium. It was concluded that ciliary contact is not necessary for generation of mucus flow provided the ciliary shear is not negated by the mucus "flake" or "slab" being in simultaneous contact with significant ciliostatic patches which would act as anchors.     

The role of the periciliary fluid in mucociliary flows: flow velocity profiles in frog palate mucus

Observations of flow velocity profiles over frog mucociliated palate are used to estimate viscosity, shear rate and shear stress in the periciliary flow field. The ability of cilia to generate significant shear stress at long distances and their utility as rhoeometers are examined. It is proposed that the depth of significant ciliary shear penetration into the periciliary fluid is sufficient to move mucus masses well beyond the ciliary tips, obviating the need for tip penetration where anchoring phenomena are sufficiently reduced.     

Cholinergic nerves stimulate mucociliary transport,ciliary activity,and mucus secretion in the frog palate

Summary Mucociliary transport, ciliary activity, and mucus secretion were studied in the palate of the frog Rana pipiens by direct observation, stroboscopic synchronization of ciliary beating, and histochemistry. Excised palates were studied in vitro, and intact palates were studied in vivo. Electrical stimulation of the glossopharyngeal nerve in vivo or of the palatine nerve in vitro stimulated all three activities. The effect was mimicked by acetylcholine and pilocarpine, enhanced by physostigmine, and blocked by atropine but unaffected by d-tubocurarine. Stimulation increased the number of cilia beating and their rate of beating, the number of goblet cells secreting and, for small acidic cells, the amount of mucus secreted, and the rate and extent of particle transport. The response to tactile stimulation was locally restricted in vitro but widespread in vivo. It was concluded that, although there is a low basal rate of mucus secretion and ciliary activity that is independent of nervous control, stimulation of these activities in the intact animal is mediated through the central nervous system and cholinergic nerves to the palate.Supported in part by Grant HL-16730 from the U.S. Public Health Service     

Mucus-depleted frog palate as a model for the study of mucociliary clearance

To better understand the frog palate model of mucociliary transport, we measured the transport rate of mucus (MTR) from the leopard frog, Rana pipiens, and from the bullfrog, R. catesbeiana, recorded the stability of the MTR over a period of hours and days and over the course of 1 yr, and measured the viscoelasticity, percent solid composition, and spinnability (filance) of mucus from both species. Bullfrog mucus was less rigid than leopard frog mucus (log G* at 1 rad/s 2.09 vs. 2.61; P less than 0.01) and had a higher viscosity-to-elasticity ratio (tan delta at 1 rad/s 0.36 vs. 0.26; P less than 0.05). It also had a lower solids content (8.71 vs. 13.72%; P = 0.02), and there was a trend to lower spinnability for bullfrog mucus (filance 26.7 vs. 33.5 mm). These data suggest that bullfrog mucus has viscoelastic properties similar to normal mammalian respiratory mucus and leopard frog mucus has viscoelasticity similar to sputum samples. MTR was significantly slower in the winter than in the summer months (17 vs. 30 mm/min; P less than 0.0001). Although the leopard frog palate could be used for at least 7 consecutive days without exhaustion, bullfrog palates could be used for only 5 days. Palates of either species could generally be tested for 6 h/day without a significant decrease in MTR. These data clarify some of the sources of variability in the use of this system and suggest methods of standardization.     

Three-dimensional imaging of the mucus secretory process in the cryofixed frog respiratory epithelium.

Using the frog palate as a representative model of human mucociliary epithelium, we analyzed, after quick freezing fixation, the three-dimensional (3-D) respiratory mucus secretory release with high voltage (200-300 kV) transmission electron microscopy (TEM). The 3-D vision of the mucus release from the secretory cells was obtained as stereo-pairs and "bas-relief" images after analysis of stereo-pairs using an image analyzer. After standard glutaraldehyde fixation, the secretory cells showed a typical goblet shape with secretory granules heterogeneous in size and electron-density which often fuse together. On the other hand, quick-frozen secretory cells exhibited a columnar shape and their membrane-bound secretory granules contained a homogeneously dark matrix. The expanded gel mucus layer was preserved and its depth never exceeded 2 microns. When the epithelium was immersed in culture medium in presence of cholinergic agonist, a marked discharge of mucus was observed and the granules swelled at the apex of the secretory cell before being discharged in the lumen. In native cryofixed epithelium, the secretory granules exhibited a marked deformability during the process of their extrusion from the secretory cell. Clusters of secretory granules surrounded by cytoplasmic material were observed in the extracellular lumen, suggesting an apocrine-type secretion. These observations indicate that rapid cryofixation and 3-D stereoscopic imaging enable a unique opportunity to analyze, without artifact, the mucous secretory process. We speculate that, apart from the classical merocrine-type secretion mechanism, the respiratory mucus may be released, at least partly by an apocrine-type secretion.     

Diffusion profiles of Na+-fluorescein in frog ventricular muscle.

Frog ventricular muscle strips were placed in a single sucrose-gap chamber to measure the interdiffusion of solutes across the sucrose-Ringer's solution partition. Steady-state diffusion profiles of fluorescein sodium developed along the axis of the muscle in the physiological node by continuously perfusing the sucrose pool with 210-mM sucrose plus fluorescein (5-10 mM). Fluorescein was found to diffuse freely through the extracellular space of the ventricular muscle without binding to the tissue. The fluorescence of Na+-fluorescein in the muscle (measured at 530 +/- 30 nm) varied linearly with the dye concentration in the sucrose perfusate. The diffusion profiles of dye in the test node depended on the tightness or snaring of the muscle strip by the latex diaphragms, the diameter of the muscle strip, and changes in hydrostatic pressure between the sucrose and Ringer's solution pools. Fluorescein concentration in the cross section of test node closest to the latex partition (sucrose-Ringer's solution interface) ranged between 4-13% of the dye concentration in the sucrose pool. These values are more than five times smaller than those estimated theoretically, assuming free diffusion. The experimental findings indicate that the presence of a physical barrier, such as a rubber diaphragm, limits free interdiffusion of solutes across the sucrose gap. The presence of such a barrier thus prevents large concentration gradients from occurring in the extracellular spaces along the physiological node.     

Stomatal mechanics. III. Geometric interpretation of the mechanical advantage*

Abstract. Previous mathematical analyses of stomatal mechanics have demonstrated, and experimental measurements have confirmed, that the turgor-generated force of the epidermal cells dominates that of the guard cells in determining aperture. DcMichele & Sharpe (1973) termed the phenomenon the mechanical advantage of the epidermal cells, while Cooke et al. (1976) expressed it as an antagonism ratio. Both of these formulations, however, have theoretical or practical limitations. This study presents a biophysical analysis demonstrating that the effective forces in the stomatal system may be studied in terms of simple stomatal geometry. From this analysis, the mechanical advantage can be redefined and interpreted based upon simple geometric relationships calculated from measurable anatomical dimensions.     

On the unmyelinated nerve glomuses in the bird's palate.

The studies on the structure and morphological variability of nerve endings in the palate mucosa of Streptopelia decaocto Friv, have been carried out. Besides single loose or dense, irregular glomuses one can observe complex glomuses as well. The former and the latter do not possess of neither capsules nor tactile cells. The author suppose that both types of glomuses can correspond to free nerve endings and receptors in which the feeling of pain is coded.     

On the role of sialic acid in the rheological properties of mucus.

The importance of sialic acid in the rheological properties of mucus has been investigated. Both bovine cervical mucus, which is a gel, and the structural glycoprotein derived from it were studied before and after treatment with neuraminidase which selectively cleaves terminal sialic acid residues. The storage modulus, viscosity and circular dichroism spectrum were all essentially changed after removal of the sialic acid. These results would indicate that removal of sialic acid does not affect the physical structure of the glycoprotein and it is concluded that sialic acid has no significant role in the rheological properties of cervical mucus.     

Transforming growth factor-beta receptor profiles of human and murine embryonic palate mesenchymal cells.

Cell signalling in the developing mammalian palate appears to involve various growth factors and hormones. An important developmental role for the transforming growth factor-beta (TGF-beta) class of growth factors is suggested by the immunolocalization of TGF-beta 1 in the palate during its ontogeny. This study examined the effects of TGF-beta stimulation of, as well as TGF-beta receptor profiles in, murine embryonic palate mesenchymal (MEPM) and human embryonic palate mesenchymal (HEPM) cells. Results showed that TGF-beta 1 (1 ng/ml) stimulated proliferation of HEPM cells and inhibited proliferation of MEPM cells in a dose-dependent manner. The time course of 125I-TGF-beta 1 binding to specific receptors was determined by incubating cells in the presence of 170 pM 125I-TGF-beta 1 for up to 4 h. In both cell types, at 37 degrees C, the binding of 125I-TGF-beta decreased linearly over 4 h, while at 4 degrees C, binding increased with time of incubation. Incubation of both cell types at 4 degrees C for 4 h, with increasing concentrations of 125I-TGF-beta 1, resulted in binding which demonstrated saturation kinetics. Scatchard analyses revealed one class of receptors for HEPM (K 32.3 pM) and MEPM (K 26.3 pM). However, SDS-PAGE analyses of 125I-TGF-beta chemically crosslinked to specific receptor sites revealed that both cell types contained the types I (65,000 Mr) and III (230,000 Mr) TGF-beta receptors while MEPM also contained the type II (86,000 Mr) receptor. Binding studies further demonstrated the ability of platelet-derived growth factor to transmodulate TGF-beta binding. These results indicate that the HEPM cell line and primary cultures of MEPM cells, although obtained from palates at similar developmental stages, are dramatically different in their responsiveness to TGF-beta and have disparate TGF-beta receptor profiles.     

On the mechanics and energetics of nectar feeding in butterflies.

A mechanistic model describing the mechanics and energetics of nectar-feeding in butterflies is developed. The butterflies Collas eurytheme and Danaus plexippus are used to illustrate the model. Simulation results indicate that there are mechanical limitations upon the range of nectar sugar concentrations and nectar extraction times available to butterflies. There is a unique optimum for net rate of energy gain at 20–25% nectar sugar concentration which is independent of the metabolic rate and of proboscis shape and size over the ranges found in butterflies. The optimal nectar extraction rate depends upon the size and shape of the proboscis. These results are discussed in relation to the design of nectar feeding structures, optimal foraging strategy, and the evolution of insect pollination.     

Development of mucociliary transport in the postnatal ferret trachea.

Little is known of the developmental aspects of mucociliary transport. Previous studies have documented that newborn ferret trachea has very few ciliated cells but numerous immature secretory cells in the epithelium and only rudimentary submucosal glands. Rapid and complete maturation occurs in the first postnatal month. This study examines mucociliary transport during this period of rapid maturation. We made direct observations of particle movement across the epithelium of ferret tracheas. No mucus transport could be demonstrated on the first day of life. Transport was discernible, although sporadic and slow, by 7 days and reached adult levels (10.7 +/- 3.7 mm/min) by 28 postnatal days. The emergence of transport capability correlated well with previously described developmental changes in ciliation, mucus secretion, and ion permeability and transport. Threshold mucus transport occurred at 1 wk of age when 20-25% of the surface cells are ciliated. The neonatal ferret appears to be a useful model for assessing integrated epithelial structure-function relationships that are important not only during early development but also during repair after airway injury involving deciliation.