QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wood, C. R. Articles by Hennessey, T. M. Search for Related Content PubMed PubMed Citation Articles by Wood, C. R. Articles by Hennessey, T. M. Social Bookmarking                         What's this?

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

Christopher R. Wood and Todd M. Hennessey^*

Dept of Biological Sciences, State University of NY at Buffalo, Buffalo, NY 14260, USA

View larger version (10K): [in a new window]   Fig. 1. Transfer of Paramecium to ß,-methylene ATP (met-ATP) causes immediate avoiding reactions (AR). Digital video imaging shows the swimming path of a cell to be relatively straight in the control solution (A), while a different cell in 25 µmol l-1 ß,-methylene ATP shows repetitive jerking back and forth, indicative of AR (B). The video capture rate was approximately 16 frames s-1. (C) The percentage of cells showing AR (% AR) increased in a concentration-dependent manner, with 100% of the cells showing AR at concentrations of ß,-methylene ATP above 25 µmol l-1 and an EC50 of approximately 12 µmol l-1. Each point represents the mean ± S.D. of three trials.

View larger version (13K): [in a new window]   Fig. 2. The electrophysiological response of Paramecium to 25 µmol l-1 ß,-methylene ATP was a transient depolarization. This is the ß,-methylene ATP-receptor potential. As the response was somewhat variable, two different cells are shown. The very fast spikes seen in the first trace (Cell 1) are the Ca2+-based, graded action potentials common to these cells but they are less evident in the second trace (Cell 2). In both traces, the cells were exposed to 25 µmol l-1 ß,-methylene ATP during the entire time. The recording medium contained 1 mmol l-1 CaCl2, 1 mmol l-1 MOPS, 0.5 mmol l-1 MgCl2 and pH 7.2 with Tris-base. The recording electrode contained 500 mmol l-1 KCl.

View larger version (16K): [in a new window]   Fig. 3. In vivo [32P]ATP binding assays were used to show saturable, external binding in the range of 1-30 nmoll-1 [32P]ATP (A). Each point represents the mean ± S.D. of at least three trials. A Scatchard plot of this data infers a single class of external [32P]ATP binding sites with an apparent Kd of approximately 23 nmoll-1 and a Bmax of 110 pmoll-1 (B). The estimate of the number of surface [32P]ATP binding sites is approximately 7.023x105 receptors cell-1.

View larger version (12K): [in a new window]   Fig. 4. Behavioral adaptation to 25 µmoll-1 ß,-methylene ATP is seen as a time-dependent decrease in ß,-methylene ATP avoidance response (% AR) (A) and in the amplitude of the ß,-methylene ATP-receptor potential (B). In both cases, it takes approximately 15 min for adaptation to be complete. Therefore, adaptation can be defined as a time-dependent loss of either the ß,-methylene ATP-AR or the ß,-methylene ATP receptor potential. Each point represents the mean ± S.D. of three trials.

View larger version (14K): [in a new window]   Fig. 5. PPNDS (pyridoxal-phosphate naphthylazo-nitro-disulfate) also caused avoidance responses (AR) and transient receptor potentials. PPNDS caused 100% AR at approximately 120 µmoll-1 and the EC50 was approximately 70 µmoll-1 (A). The transient receptor potential (B) caused by 100 µmoll-1 PPNDS was similar in amplitude and duration to those seen with 25 µmoll-1 ß,-methylene ATP (compare with Fig. 2). Two different cells are shown to demonstrate the variability of the responses. The amplitude of the PPNDS receptor potential decreased as a function of time of exposure to 100 µmoll-1 PPNDS (C). The behavioral bioassays and the recording solution were the same as the solutions used for Figs 1, 2. Each point represents the mean ± S.D. of three trials.

View larger version (15K): [in a new window]   Fig. 6. Behavioral cross-adaptation seen as a function of time in either 25 µmol l-1 ß,-methylene ATP (met-ATP) or 100 µmol l-1 PPNDS (pyridoxal-phosphate naphthylazo-nitro-disulfate). Cross-adaptation between two ligands is seen as the loss of responsiveness to one ligand following exposure to the other ligand and vice versa. (A) Cells were adapted to 25 µmol l-1 ß,-methylene ATP and then retested in either 25 µmol l-1 ß,-methylene ATP (open circles) or 100 µmol l-1 PPNDS (filled circles). Cross-adaptation was seen in response to either ligand with similar time courses. (B) Cross-adaptation was also demonstrated by incubation in 100 µmol l-1 PPNDS and retesting in either 25 µmol l-1 ß,-methylene ATP (open circles) or 100 µmol l-1 PPNDS (filled circles). Each point represents the mean ± S.D. of three trials.

View larger version (28K): [in a new window]   Fig. 7. Behavioral adaptation to either 100 µmol l-1 PPNDS (pyridoxal-phosphate naphthylazo-nitro-disulfate) or 25 µmol l-1 ß,-methylene ATP is associated with a reversible loss of external [32P]ATP binding. (A) When assayed as a function of time, the % [32P]ATP bound to live cells decreased over 15 min exposure to 100 µmol l-1 PPNDS. (B) When the % [32P]ATP bound to live cells was assayed after a 15 min exposure to either 25 µmol l-1 ß,-methylene ATP or 100 µmol l-1 PPNDS, both caused dramatic decreases in [32P]ATP binding. When cells were adapted to either 25 µmol l-1 ß,-methylene ATP or 100 µmol l-1 PPNDS, washed free of the adapting ligand and then `de-adapted' in wash buffer for 15 min, the [32P]ATP binding returned to normal levels. This showed that the adaptation was reversible. Each point represents the mean ± S.D. of three trials.

View larger version (36K): [in a new window]   Fig. 8. The amount of [32P]ATP bound to live cells was reduced dramatically by including a 50-fold excess of either ß,-methylene ATP (met-ATP) or PPNDS (pyridoxal-phosphate naphthylazo-nitro-disulfate) in the binding assay. The control column represents the amount of [32P]ATP bound in the absence of any extra added ligand, and each of the other columns is normalized to those counts. Each point represents the mean + S.D. of three trials.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter