In vitro studies on the regulatory mechanisms of ciliary activity of frog palate epithelium

The regulatory mechanisms of ciliary activity of frog palate epithelial cells are investigated in this study. Ciliary responses to electrical, mechanical and autonomic neurotransmitter are characterized with measuring ciliary frequency at single cell level. Ciliated cells of frog palate outgrowth cultures respond to electrical, mechanical and acetylcholine stimuli by an increase of ciliary beat frequency. The ciliary beat frequency shows dose response to electrical stimulation. Under effective electrical stimulation ciliary beat frequency increase is transmitted cell to cell from explant to outgrowth at a speed of 1.8 cells/second. Under mechanical stimulation ciliary response is transmitted in the same way as with electrical stimulation, but from stimulated cell to adjacent cells in all directions at a speed of 1.4 cells/second. Ciliated cells do not respond to epinephrine, but respond to acetylcholine in a dose dependent manner. The ciliary response to acetylcholine is inhibited specifically by atropine. Direct innervation of frog palate ciliated cells has been demonstrated in this study with electron microscopy. Electrical stimulation of the palate outgrowth culture can induce ciliary beat frequency increase of ciliated cells in the culture. This ciliary response results from palatine nerve endings in the explant releasing acetylcholine in response to the stimulation. Either removal of explant from a culture or atropine treatment inhibits outgrowth ciliated cells from responding to electrical stimulation. The morphology and ciliary activity of the palate outgrowth cells over three weeks in culture are characterized. Outgrowth of the primary culture is formed by migration of ciliated epithelium from the explant. The outgrowth maintains stratified epithelial characteristics of the palate epithelium, but secretory granule in basal secretory cells are gradually lost in culture. As a coincidence ciliary beat frequency gradually increases. The ciliary beat pattern changes from frequent fluctuation to a relative constant frequency with culture age. It is suggested that the basal secretory cells of the palate may inhibit ciliary activity by releasing secretory granules. Gap junctions are found between epithelial cells in both explants and outgrowths. Intercellular communication by gap junction provides an integrated control mechanism for regulation of the ciliary activity of large numbers of cells in the epithelium. Stimulation of a single cell or a few cells induces ciliary activity in many cells. Mechanical stimulation and gap junctional intercellular communication may represent a mechanism of local activation of mucociliary transport in response to exogenous particles. Electrical stimulation and gap junctional intercellular communication provides a mechanism of more general activation of mucociliary transport mediated by the palatine nerve. The effect of Ca²⁺ in mediation of ciliary response to stimulation is examined. It is found that extracellular Ca²⁺ is not necessary for initiation of ciliary response to a stimulation, but is important in maintaining the stimulated ciliary activity. It is suggested that mitochondria and nuclei are the major intracellular Ca²⁺ storage sites. Mobilization of Ca²⁺ from mitochondria and nuclei into the cytoplasm and cilia initiates ciliary beat frequency increase to various stimulations. Free Ca²⁺ or other signal molecules in stimulated cells passing through gap junctions into adjacent cells may cause the propagation of the ciliary response.