External GTP alters the motility and elicits an oscillating membrane depolarization in Paramecium tetraurelia.

Abstract

Paramecium, a unicellular ciliated protist, alters its motility in response to various stimuli. Externally added GTP transiently induced alternating forward and backward swimming interspersed with whirling at a concentration as low as 0.1 jFM. ATP was 1000-fold less active, whereas CTP and UTP produced essentially no response. The response to the nonhydrolyzable GTP analogs guanosine 5'-[y-thio]triphosphate and guanosine 5'-[13,y-imido]triphosphate was indistinguishable from that to GTP. This behavioral response was correlated with an unusual transient and oscillating membrane depolarization in both wild-type cells and the mutant pawn B, which is defective in the voltage-dependent Ca2+ current required for action potentials. This is a specific effect of external GTP on the excitability of a eukaryotic cell and, to our knowledge, is the first purinergic effect to be discovered in a microorganism.Paramecium tetraurelia normally swims forward except for occasional brief periods of backward swimming or whirling, a randomly directed motion (1). However, many stimuli, thermal, electrical, mechanical, or chemical, can alter the swimming speed and the frequency and duration of backward swimming and whirling events (2-6). These responses are normally transient. Cells return to their prestimulus behavior even in the continued presence of the stimulus, a form of sensory adaptation. The combination of behavioral response and subsequent adaptation can result in attraction to or repulsion from a stimulus (6). These swimming behaviors generally have clear, well-studied, and readily measurable electrophysiological correlates that can aid in unraveling a signal-transduction pathway. For example, increased swimming speed is correlated with membrane hyperpolarization, whereas decreased swimming speed is correlated with membrane depolarization. Strong depolarizations can elicit graded Ca2+-based action potentials, resulting in whirling and backward swimming due to increased intraciliary Ca2+ (3,4).To quantify these swimming behaviors, we have developed a computerized motion analysis assay that measures the percentage of total path time spent whirling and undergoing transitions between forward and backward swimming [defined as percent directional changes (PDCs) (1)]. While using this assay to quantify the behavioral effects of externally added nucleotides, we found that guanine nucleotides specifically and potently altered the swimming behavior of paramecia. In addition, while attempting to corroborate the GTP-induced behavior of the cell with changes in membrane potential, we discovered an electrophysiological response.

Oxidants act as chemorepellents in Paramecium by stimulating an electrogenic plasma membrane reductase activity

“…This type of response is also seen with other depolarizing stimuli such as heat (Hennessey et al 1983), anterior mechanical stimulation (Machemer 1988), ionic stimulation (Saimi and Kung 1987) or other chemorepellents such as quinine (Van Houten 1979) and GTP (Clark et al 1993).…”

“…Since the extent of attraction is dependent upon the membrane potential, repellent-induced depolarizations may modify attractant responses. For example, neutrophil attraction is inhibited by external GTP (Boonen et al 1991), a known chemorepellent in Paramecium (Clark et al 1993).…”

PPNDS is an agonist, not an antagonist, for the ATP receptor of Paramecium

“…6B). By contrast, cells that were behaviorally adapted to β,γ-methylene ATP or PPNDS still retained full responsiveness to another non-toxic, depolarizing chemorepellent, 10 µmol l -1 GTP (Clark et al, 1992). These adapted cells also showed normal AR in the standard ionic depolarizing test solutions (Saimi and Kung, 1987) such as 40 mmol l -1 K + , 10 mmol l -1 Na + and 8 mmol l -1 Ba 2+ .…”

Ca2+ oscillations mediated by exogenous GTP in Paramecium cells: assessment of possible Ca2+ sources

“…In Paramecium the discovery has been made that exogenous guanosine trisphosphate, GTP, causes periodic back-and forward swimming, paralleled by oscillating Ca 2+ currents [22]. This observation has been extended to the related species, Tetrahymena [23].…”

“…It suggests involvement of somatic channels or of ciliary channels of another type which is not known as yet. While this has been known from pawn cells already [22], the involvement of any Ca 2+ channels has been questioned altogether [41]. To us, involvement of some somatic Ca 2+ channels appears appealing, not only because we see a Ca 2+ influx component (Fig.…”