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     

Calcium and the regulation of mammalian ciliary beating

Summary This report summarizes our recent work on the role of intracellular Ca²⁺ ([Ca²⁺]_i) in regulating mammalian ciliary beat frequency (CBF). CBF from a single ovine cilium and [Ca²⁺]_i from the same cell were measured by digital video phase contrast microscopy and fura-2 ratiometric imaging video microscopy, respectively. Cells were stimulated with two exposures to 10 M acetylcholine (ACh). CBF was recorded during the first and [Ca²⁺]_i during the second stimulation. ACh increased [Ca²⁺]_i and CBF transiently with indistinguishable kinetics and, early in culture, even induced [Ca²⁺]_i oscillations and ciliary frequency modulations with the same peak-to-peak time interval. Cells treated with 1 M thapsigargin, an inhibitor of the endoplasmic-reticulum Ca²⁺-ATPase, showed transient [Ca²⁺]_i and CBF increases, again with similar kinetics, which often remained at an elevated plateau. Application of ACh to cells pretreated with thapsigargin produced decreases in both [Ca²⁺]_i and CBF. Finally, changing extracellular Ca²⁺-concentrations induced corresponding changes in [Ca²⁺]_i that were associated with kinetically similar CBF changes. These data strongly suggested that [Ca²⁺]_i is a critical signal to regulate CBF in mammalian tracheal epithelial cells. In an initial effort to provide constraints on the number and type of reactions that link changes in [Ca²⁺]_i to changes in CBF, simultaneous recordings of both signals from a single cell were analyzed. Such recordings provided higher resolution of the kinetic responses of CBF and [Ca²⁺]_i to ACh as well as they allowed direct assessment of the coupling between [Ca²⁺]_i and CBF. Simultaneous measurements revealed that [Ca²⁺]_i and CBF were perfectly correlated within the CBF measurement time resolution, except for the period of the fastest changes in both signals during the initial ACh exposure. There, changes in CBF lagged the changes in [Ca²⁺]_i by 1–3 ciliary beat cycles (ca. 150–450 ms).     

On different types of nerve endings in the frog median eminence after fixation with permanganate and glutaraldehyde-osmium

Summary In the frog median eminence, fixed with glutaraldehyde and osmium tetroxide, four types of nerve endings can be generally distinguished. These endings are in contact with the pericapillary spaces of primary portal vessels and can be identified by the internal structure and the size of their granules and vesicles. Type 1 contains large granules (1500–2400 Å in diameter) and small clear vesicles (300–500 Å in diameter), type 2 intermediate granules (about 1100–1700 Å in diameter) and small clear vesicles, type 3 small granules (about 600–1000 Å in diameter) and small clear vesicles, type 4 only numerous small clear vesicles. The mixed types containing the large, intermediate and small dense granules in the same ending are infrequently found.After KMnO₄ or LiMnO₄ fixation the granules and vesicles mentioned above are observed as follows. The large granules in the type 1 nerve ending appear mostly pale or less-dense. The intermediate granules in the type 2 also appear mostly pale or less-dense, but some frequently show granules of high density. The small granules in the type 3 consistently contain the dense substance and these endings can be subdivided into two different types according to the populations of different sizes of dense granules [type 3a (900–1000 Å) and type 3b (500–800 Å)]. Dense-cored and cleared-synaptic vesicles are frequently present with together in the type 3 endings. The small vesicles (300–400 Å), in the type 4, appear generally pale (type 4a), but some nerve endings contain small dense cored-vesicles (type 4b).The author wishes to thank Prof. H. Fujita for his advice and criticism.     

On the uptake of exogenous catecholamines by adrenal chromaffin cells and nerve endings

Summary Light-microscopic autoradiography has revealed characteristic labelling patterns in adrenal medullary cells following the intravenous administration of different catecholamines. The uptake patterns for [³H] dopa, [³H] dopamine, [³H] noradrenaline and [³H] adrenaline have been compared. In all cases A cells were more active than NA cells and cells situated in the zone nearest the cortex demonstrated a markedly higher rate of uptake than central cells. It was concluded that adjacent chromaffin cells with very similar morphology may differ as much as 50 fold in their capacities to incorporate exogenous amines. The adrenergic nature of the innervation of the vessels of the adrenal cortex and capsule in the mouse was confirmed.     

The effects of niamid and reserpine on the nerve endings of the pineal gland

Summary Kitten pineal glands were studied cytochemically under normal conditions, after reserpine injection, and after niamid administration. Adrenergic nerve elements were in perivascular spaces while cholinergic terminals were adjacent to pinealocytes, often times in synaptic contact. BA reactions are primarily in dotted vesicles of adrenergic terminals with some reaction in granular vesicles. Positive reaction occurs along neurotubules and membranous structures of adrenergig nerve fibers and terminals indicating membrane-bounded BA's. Niamid increased the number and density of dotted vesicles, and some granular vesicles are increased in density and size. Reserpine produced a loss reaction in dotted vesicles and a loss of vesicle matrix, producing elliptical vesicles. There is loss of reaction of the dotted vesicles, but occasionally, the positive granular reaction remains. Cholinergic terminals demonstrate no changes with either niamid or reserpine. These findings indicate BAs are stored in reserpine sensitive dotted vesicles and membraneous structures. The findings also show that the dotted vesicle matrix is reserpine sensitive and is necessary for storage of the BA's. Possibly biogenic amines cannot be stored or synthesized in terminals unless the matrix of the dotted vesicle is intact.Supported by: HEW Grant No. NS-10326. The University of Texas Medical School at Houston. — Special appreciation to Mrs. Charlotte Smith for her valuable technical assistance. Appreciation to Ciba-Geigy Corporation for supply of Serpasil (reserpine).     

Muscle cells in a nerve trunk of a frog muscle

Summary Three muscle fibers were identified by electron microscopy within a nerve of a frog muscle. They resembled extrafusal muscle fibers but were located in an endoneurial rather than in an endomysial compartment. To call these endoneurial muscle fibers the obvious continuation of extrafusal fibers of a muscle spindle is certainly unwarranted; to label these fibers ectopic and to let the matter rest there is probably an understatement of sorts.     

Ultrastructure of free nerve endings in respiratory and squamous epithelium on the rat nasal septum

The distribution of nerve fibres in the mucosa of the nasal septum of the rat was investigated by means of transmission electron microscopy on transverse and tangential ultrathin sections. Near the basement membrane of respiratory and squamous epithelium, a rather dense network of unmyelinated nerve fibres occurs. Some fibres in the respiratory epithelium ascend between the epithelial cells to reach up to the tight junctions. These fibres appeared in transverse sections to end as hooks or boutons, sometimes with branches. These shapes resemble the free nerve endings that are considered to act as nociceptors. The small intraepithelial fibres, with diameters of about 0.5–1 m, contain both dense granules and clear vesicles comparable to synaptic vesicles. Substance P was found in dense granules in basal fibres; vasoactive intestinal peptide was absent throughout the epithelium. Acetylcholinesterase activity was observed closely associated with the basal fibres; the apical fibres showed little if any activity. Membrane specializations pointing to an efferent function as well as structures usually associated with mechanoreceptive functions were lacking in both respiratory and squamous epithelium.Part of this work was presented at the Annual Conference of the Netherlands Society for Electron Microscopy, 28–29 Nov 1985, Wageningen, The Netherlands. See Spit BJ and Hendriksen EGJ (1986) Ultramicroscopy 19:102–103     

Structure of cells and nerve endings in abdominal vagal paraganglia of the rat

Summary The abdominal vagal paraganglia of the rat consist of small groups of cells, interspersed by blood vessels and nerve bundles and lying close to, or within, the vagus nerve or its branches. Each cell group consists of 2–10 Type I cells incompletely invested by 1–3 satellite cells. Type I cells are characterised by the presence of numerous dense-cored vesicles in their cytoplasm and may exhibit synaptic-like contact with each other.Small efferent nerve endings make synaptic contacts with Type I cells. Larger cup-shaped afferent nerve endings also make synaptic contacts of two kinds with Type I cells. Nerve-nerve synapses are often seen within or close to paraganglia.Attention is drawn to the close similarity of fine structure of abdominal vagal paraganglia, carotid body and small intensely fluorescent cells of the superior cervical ganglion in rats. Possible functional implications of this morphological similarity are discussed.     

Mass transport at rotating disk electrodes: effects of synthetic particles and nerve endings

An unstirred layer (USL) exists at the interface of solids with solutions. Thus, the particles in brain tissue preparations possess a USL as well as at the surface of a rotating disk electrode (RDE) used to measure chemical fluxes. Time constraints for observing biological kinetics based on estimated thicknesses of USLs at the membrane surface in real samples of nerve endings were estimated. Liposomes, silica, and Sephadex were used separately to model the tissue preparation particles. Within a solution stirred by the RDE, both diffusion and hydrodynamic boundary layers are formed. It was observed that the number and size of particles decreased the following: the apparent diffusion coefficient excluding Sephadex, boundary layer thicknesses excluding silica, sensitivity excluding diluted liposomes (in agreement with results from other laboratories), limiting current potentially due to an increase in the path distance, and mixing time. They have no effect on the detection limit (6 ± 2 nM). The RDE kinetically resolves transmembrane transport with a timing of approximately 30 ms.     

Development and ciliation of the palate in two frogs, Bombina and Xenopus; a comparative study

The morphological changes that occur during metamorphosis in the palates of two types of anuran larvae (a discoglossid, Bombina orientalis, and a pipid, Xenopus laevis) are compared. In B. orientalis the structural changes are accompanied by the ciliation of the palate epithelium. Ciliation begins in the anterior region of the palate and continues in a posterior direction throughout metamorphosis. By contrast, the palate of X. laevis never becomes ciliated during its development. Instead, two ciliated grooves develop between the choanae (nasal openings) and the esophageal opening. The grooves transport mucus and trapped objects out of the internal nares and toward the esophagus. These grooves are compared to similar structures on the palate of adult B. orientalis. The timing and pattern of ciliogenesis during metamorphosis in each of these anurans is also described relative to well-established staging series for external frog development. We show that the onset and location of ciliogenesis are consistent and predictable in these anurans and, therefore, make the frog palate an excellent system for the study of ciliogenesis.     

Regulation of outer-arm-dynein activity by phosphorylation and control of ciliary beat frequency

Summary Ciliary beating is empowered by a mechanochemical enzyme, dynein, which appears as two rows of projections on doublet microtubules. While inner-arm dyneins modulate beat form, outer-arm dynein empowers ciliary beat and sets beat frequency. Beat frequency is controlled via phosphorylation of outer-arm dynein. UsingParamecium tetraurelia as model system, we have previously identified a regulatory light chain of outer-arm dynein (22S dynein), M_r29 (p29), whose phosphorylation is cAMP-dependent. The phosphorylation state of the p29 in 22 S dynein determines in vitro microtubule translocation velocity. Although in vitro phosphorylation of p29 takes place in a short time, the percent change ist significantly less than the percent change in dynein activation, or in ciliary beat frequency. A potential mechanism that explains how a few activated dyneins can change ciliary beating is discussed.     

Fine structure,origin, and distribution density of the autonomic nerve endings in the tarsal muscle in the eyelid of the mouse

Summary The fine structure, origin, and distribution density of the autonomic nerve endings in the tarsal muscle of the mouse were studied by histochemistry and electron microscopy. With histochemical methods, the fine nerve plexus in the normal muscle shows both catecholamine-positive varicose fibers and acetylcholinesterase-active varicose fibers. The former are distributed more densely than the latter. After superior cervical ganglionectomy, the catecholamine-positive fibers disappear, while after pterygopalatine ganglionectomy, the acetylcholinesterase-active fibers vanish. In electron micrographs, the varicosities appear as expansions containing many synaptic vesicles. The axonal expansions partly lack a Schwann sheath and directly face the pinocytotic vesicle-rich zones of the smooth muscle cells. A relatively wide space, 0.1 to 1.0 m in width, lies between nerve expansion and muscle cell. The expansions can be classified into two types: Type I having small granular synaptic vesicles, and Type II having agranular vesicles instead of small granular synaptic vesicles. Type I undergoes degeneration after superior cervical ganglionectomy, while Type II degenerates after pterygopalatine ganglionectomy. This indicates that Type I corresponds to the synaptic ending of the adrenergic fiber originating from the superior cervical ganglion, and Type II to the synaptic ending of the cholinergic nerve fiber derived from the pterygopalatine ganglion. Type I is more frequent (88/10⁴ m² area of muscle) than Type II (17/10⁴ m²).     

The ciliary basal apparatus is adapted to the structure and mechanics of the epithelium

The basal apparatuses which anchor the gill cilia in Branchiostoma lanceolatum (Pallas) and the actinopharynx cilia in Calliactis parasitica (Couch) are similar in structure. In C. parasitica the pharynx epithelium and the basal apparatuses are flexible. The basal apparatuses, however, bend in only one direction. This mechanism may permit epithelial flexibility whilst maintaining a similar basal orientation between cilia. In B. lanceolatum the ciliated gill epithelia are mechanically stable but the epithelial surfaces are curved. The basal apparatuses may correct for this curvature, with short rootlets between the distal centrioles (basal bodies) and the cell membranes, so that their cilia also share a common orientation. A common basal orientation between cilia is important for their coordination. The degree of coordination depends upon the function of the cilia; water-propelling cilia are more precisely coordinated than mucus-propelling cilia. Much of the structural diversity of ciliary basal apparatuses in Metazoa may be due to variation in the demands of anchoring functionally different cilia to epithelia which have different structural and mechanical properties.     

Structure and distribution of gap junctions in lens epithelium and fiber cells

Summary We report a comparative study of gap junctions in lens epithelia of frog, rabbit, rat and human, using a double mounting method for freeze-fracture electron microscopy. The gap junctions on the narrow sides of hexagonal cortical fiber cells of various species were also studied with the same technique. Gap junctions were commonly present between epithelial cells of the entire undifferentiated epithelium, between fiber cells on both wide and narrow sides, and between epithelial cells and fiber cells. Structural diversity of gap junctions, based on connexon arrangements, was evident in lens epithelia among the four species studied. Gap junctions with random arrays of connexons were found predominantly in frog lens epithelium, while the crystalline and striated configurations were mainly observed in the epithelia of human and rat, and of rabbit, respectively. On the other hand, there was no structural variation of gap junctions observed on either wide or narrow sides of lens fiber cells from any species studied. Only the random-type gap junction was found. However, the distribution of gap junctions was unique on the narrow sides. There was a single row of junctional plaques along the middle of the narrow sides, whereas the wide sides showed an uneven distribution pattern. The gap junctions between epithelial cells and fiber cells had a random packing of connexons.     

Internalized gap junctions in ciliary epithelium of rabbit and rat

Summary Transmission electron microscopy was used to examine internalized gap junctions (IGJ) in rabbit and rat ciliary epithelial cells. A prominent feature of all the specimens studied was the presence of different images of IGJ membrane that entrapped a portion of an adjoining cell. We documented and analyzed more than 500 gap junction (GJ) vacuoles and invaginations, the latter comprising less than 20% of all the structures examined. With ten exceptions found in non-pigmented cells, all the IGJ were unidirectionally internalized within the cytoplasm of pigmented epithelial cells. Morphological signs of autophagic degradation of GJ vacuoles were observed. An essential finding was that once a GJ membrane started to invaginate, a lucidation of a part of the protruding cytoplasm occurred; no planar GJ membranes exhibited such an alteration. The present findings suggest that IGJ derived from the epithelium of ciliary processes arise through an invagination-endocytosis mechanism and are degraded autophagically. This phenomenon may be relevant to aqueous humor production.     

Bisphenol A inhibits compound action potentials in the frog sciatic nerve in a manner independent of estrogen receptors

Although the endocrine disruptor bisphenol A (BPA) is reported to inhibit nerve conduction, the underlying mechanisms are unclear. Therefore, in the present study, we examined the effect of BPA on compound action potentials (CAPs) recorded from the frog sciatic nerve using the air-gap method. Treatment of the sciatic nerve with BPA (0.5 mM) for 20 min reduced the peak amplitude of the CAP by approximately 60% in a partially reversible manner. The reduction in the CAP peak amplitude was concentration-dependent, with a half-maximal inhibitory concentration (IC₅₀) value of 0.31 mM. This effect of BPA was unaffected by an estrogen-receptor antagonist, 4-hydroxytamoxifen, which by itself reduced CAP peak amplitude, with an IC₅₀ value of 0.26 mM (comparable to that of BPA). The natural estrogen 17β-estradiol, at the highest dissolvable concentration (0.05 mM), had an effect similar to that of BPA. The IC₅₀ value of BPA was comparable to those of some local anesthetics in inhibiting frog CAPs. Our findings suggest that BPA inhibits nerve conduction in a manner independent of estrogen receptors. This action of BPA may underlie, at least in part, the neurotoxicity of the compound.     

Calcium and ATP regulation of ion transport in larval frog skin

Ion transport measured as short circuit current (Isc) across the skin of larval frogs is activated by amiloride, acetylcholine, and ATP. In many epithelia, ATP stimulation of Isc involves an increase in intracellular calcium. To define the role of changes in intracellular calcium in ATP stimulation of Isc in larval frog skin, epithelial cells were loaded with calcium by adding 5 μM ionomycin to a 2 mM calcium apical Ringer's solution. Calcium loading had no observable effect on baseline Isc or on stimulation by ATP. Minimizing changes in intracellular calcium by loading the cell with the calcium chelator BAPTA also had no measurable effect on ATP stimulation of Isc. When the apical side was bathed with Ca²⁺-free Ringer's solution, ionomycin increased Isc up to 15 μA. This increase was partially blocked by 2 mM Ca²⁺, 2 mM Mg²⁺, and 10 μM W-7. Other experiments showed that baseline-stimulated and ATP-stimulated Isc were always larger in 2 mM Mg²⁺ Ringer's compared to 2 mM Ca²⁺. In dissociated cells bathed in 2 mM Ca²⁺ Ringer's, ATP had no effect on intracellular calcium as measured by Fluo-LR fluorescence changes. In conclusion, ATP apparently stimulates Isc without concomitant changes in intracellular calcium. This is consistent with a directly ligand-gated receptor at the apical membrane with P2X-like characteristics. Accepted: 21 April 1999     

Presynaptic plasma membranes and synaptic vesicles of cholinergic nerve endings demonstrated by means of specific antisera

Summary Antisera were raised to cholinergic presynaptic plasma membranes and synaptic vesicles isolated from the electric organ of Torpedo marmorata and tested by immunochemical and immunohistochemical methods. The antisera responded to many antigens not specific to nerve endings, but it was possible to eliminate these antibodies by means of simple absorption procedures with fractions containing the unwanted antigens. After absorption, staining of thin sections of electric organ by immunofluorescence was limited to the region of nerve endings in the tissue.The remaining antibodies responded in the case of the plasma membrane antisera predominantly to a 33,000 molecular-weight polypeptide and a chloroform/methanol-soluble antigen. In cross reactivity studies it was found that this antiserum not only stains cholinergic nerve endings in Torpedo but also those in mammalian tissue. The antigen responsible for the cross reactivity is restricted to the chloroform/methanol-soluble material.The vesicle antiserum labels cholinergic nerve endings in mammalian tissue as well; the relevant antigen in this case is different from the one described above and is likely to be a glycosaminoglycan. The antisera provide valuable markers for cholinergic nerve terminals. In addition, the vesicle antiserum may now be used to study axonal transport and the life cycle of this organelle in the cholinergic neurone.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EGTA ethylenebis (oxoethylenenitrilo) tetra-acetic acid - MW apparent molecular weight Enzymes. Na⁺, K⁺-activated ATPase (EC 3.6.1.3); acetylcholine esterase (EC 3.1.1.7); choline acetyl-transferase (EC 2.3.1.6)