Trifluoperazine‐induced changes in swimming behavior of paramecium: Evidence for two sites of drug action

Abstract

Trifluoperazine (TFP), a drug that binds to Ca2+-calmodulin (CaM) complexes, altered swimming behavior not only in living paramecia, but also in reactivated, Triton-extracted "models" of the ciliate. By comparing the responses of living cells and models, we have ascertained that two sites of drug action exist in paramecium cilia. Swimming movements were recorded in darkfield stroboscopic flash photomicrographs; this permitted accurate quantitation of velocities and body-shape parameters. When living paramecia were incubated in a standard buffer containing 10 microM TFP, their speed of forward swimming fell over several minutes and their bodies shortened. Untreated paramecia backed up repeatedly and frequently upon transfer to a solution containing barium ions (the "barium dance"), but cells preincubated in TFP did not "dance." Instead they swam forward slowly for long periods of time without reversing and occasionally then exhibited abnormally prolonged reversals. W7 effects on swimming mimicked low doses of TFP, and the analog W5 did not visibly alter normal swimming patterns. These results suggest that TFP induces a decrease in the intracellular pCa of living paramecia, perhaps by reducing the efficiency of a calmodulin-activated calcium pump in the cell membrane. Paramecia extracted with Triton X-100 and reactivated to swim forward (7 greater than or equal to pCa greater than or equal to 6) were not affected by addition of up to 40 microM TFP to the reactivation medium. We conclude that the main drug effect in living cells is probably not at the axoneme. However, at low pCa, TFP directly affected the ciliary axoneme to shift its behavior to one characteristic of a higher pCa: TFP inhibited backward swimming in models reactivated at pCa less than 6; instead they swam forward or rocked in place. The mechanism of ciliary reversal in paramecium may therefore depend on an axonemal Ca2+-sensor, possibly bound CaM, which is affected by TFP only at low pCa, as has been postulated for other types of cilia.

Calcium sensitivity extends the length of ATP-reactivated ciliary axonemes.

“…Studies using calmodulin antagonists with reactivated models indicate that calmodulin plays a role in triggering Ca-dependent ciliary activation (12,29), as well as other Ca-mediated axonemal responses (12,(30)(31)(32)(33)(34). In addition, fluorescence and immunoelectron microscopy show that calmodulin is distributed along the entire length of whole cilia (35) and outer-doublet microtubules (36).…”

A calcium regenerative potential controlling ciliary reversal is propagated along the length of ctenophore comb plates.

“…The length constant for a cilium of "infinite"length has been calculated to be as high as one to several millimeters (4, 31), indicating that the short ,um) cilia of protozoa are essentially isopotential along their length and with the cell body (7,32 (12)(13)(14)(15). Fluorescence and immunoelectron microscopy have shown that calmodulin is localized along the entire length of the axoneme (16,17 Therefore, our finding that Ca2+ influx occurs over most of the length of comb-plate cilia implies that the Ca2+-sensitive machinery controlling beat direction is also distributed along the length of the axoneme.…”

“…Calmodulin, a putative ciliary Ca2+ sensor (12)(13)(14)(15), has been detected along the entire length of cilia (16,17). In contrast, local application of Ca2+ to reactivated cilia and flagella indicate that the Ca2+-sensitive machinery controlling various motor responses is localized to a specific region of the axoneme (18)(19)(20).…”

Ciliary reorientation is evoked by a rise in calcium level over the entire cilium

“…In addition, calmodulin probably activates membrane Ca 2ϩ pumps that lower intraciliary Ca 2ϩ to restore forward beat [Otter et al, 1984]. In the axoneme of Chlamydomonas, calmodulin is found near the bases of the radial spokes and also in the sheath surrounding the central microtubules, positions that could be important for switching of ciliary activity during reversal [Yang et al, 2001].…”