J. Membrane Biol. 127, 185-193 (1992) The Journal of Membrane Bioloffy 9 Springer-Verlag New York lnc, 1992 Possible Mechanism of Ciliary Stimulation by Extracellular ATP: Involvement of Calcium-Dependent Potassium Channels and Exogenous Ca 2+ T. Weiss?, L. Gheber:~, V. Shoshan-Barmatz+, and Z. Priel$ ~-Department of Life Sciences and :)Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, 84105 Israel Summary. Ciliary motility was examined optically in tissue cul- tures from frog palate epithelium and frog's esophagus as a func- tion of extracellular concentration of adenosine 5'-triphosphate (ATP) and related compounds. The addition of micromolar con- centration of ATP caused a strong enhancement of frequency and wave velocity in the direction of the effective stroke. Since adenosine 5'-[/~,y imido]-triphosphate (AMP-PNP), a nonhydro- lyzable analog of ATP, produces the same effects, ATP hydrolysis is not required. The overall potency is ATP ~ AMP-PNP > ADP > adenosine > AMP. It is suggested that both the phosphate and the base moieties are involved in ATP binding. The enhancement of ciliary activity by extracellular ATP is dependent on the presence of extracellular Ca 2., which can be replaced by extracellular Mg>. The effect of a number of potent inhibitors of the voltage-gated calcium channels on the stimula- tion of ciliary activity by ATP were examined. No effect was detected in the concentration range within which these agents are specific. On the other hand, quinidine, a potent inhibitor of K § (calcium-dependent) channels, inhibits the effect of ATP. The following model is suggested: exogenous ATP interacts with a membrane receptor in the presence of Ca >, a cascade of events occurs which mobilizes intracellular calcium, thereby increasing the cytosolic free Ca 2+ concentration which conse- quently opens the calcium-activated K" channels, which then leads to a change in membrane potential. The ciliary response to these changes is the enhancement of ciliary activity. Key Words extracellular ATP . ciliary activity . K:(Ca 2.) channels - quinidine 9 Ca 2- channels Introduction Cilia are densely packed cellular protrusions, num- bering 100-200 per cell. Their main function is trans- port of water or mucus. In order to fulfill this task, cilia perform synchronous periodic beating and form a wave in space and in time, called the metachronal wave. Cilia are divided into cilia which propel water and cilia which propel mucus (a "coat" of viscoelas- tic fluid 1-30/xm in thickness which covers the cili- ated epithelium). The water-propelling cilia are found in ciliated protozoa and in molluscs, are rela- tively long (10-30/xm) and are not densely packed. Mucus-transporting cilia occur in respiratory, repro- ductive and digestive systems of mammals and am- phibians. They are short (5-7 /xm) and densely packed, and as a result, they are quite difficult to investigate. Ciliary cells are excitable cells in the sense that they can respond to a variety of stimuli, mechanical, electrical, chemical and hormonal, by altering the pattern of the ciliary activity. In water- propelling cilia, a systematic study of the influence of the membrane potential on the ciliary activity was performed (Andrivon, 1988; Machemer, 1990). In addition the direct influence of Ca 2+ ions on the pattern and the frequency of the ciliary beating of demembranated protozoa was investigated (Naitoh & Kaneko, 1973). It is now accepted that these two factors play an important role in controlling the cili- ary activity of water-propelling cilia. With mucus- transporting cilia little work was done mainly be- cause of experimental difficulties. There is evidence that the membrane potential or the intracellular con- centration of Ca 2+ participate in controlling ciliary activity (Eckert, 1972; Eckert & Murakami, 1972; Verdugo, 1979; Sanderson & Dirksen, 1986; Vil- lalon, Hinds & Verdugo, 1989). Questions such as whether the membrane potential and the intracellu- lar calcium ion concentration are coupled or inde- pendent and what is their relative importance or whether the extracellular calcium or calcium from intracellular storage is used are still to be answered. The pronounced effect of extracellular ATP on ciliary transport rate and frequency has been known for a long time (Varhaus & Deyrup, 1953; Usuki, 1959; Murakami, Machemer-Rohnisch & Eckert, 1974; Nelson & Wright, 1974; Ovadyahu, Eshel & Priel, 1988; Villalon et al., 1989). As faras we know, this is the strongest effect produced by any extracel- lular compound on ciliary beating. The concentra- tion of ATP required to increase the ciliary fre- quency is in the range of 10 to I00 /XM, a feasible