(IBMX is added to decrease phosphodiesterase activity. In general, early stationary stage cells were harvested by centrifugation and washed by centrifugation in basic buffer: 1 mM CaCl 2, 1 mM MOPS (3- propane sulfonic acid), 1 mM IBMX (1-isobutyl-1-methyl xanthine), 0.01 mM EDTA with various salts indicated, adjusted to pH 7.2 with Tris base. The attractant responses are consistent and robust, as shown in this paper. tetraurelia, albeit at higher concentrations than used by Preston. Likewise, in our studies, glutamate is an attractant to P. Preston and Usherwood (1988) described attraction to glutamate and specific binding sites on cilia. Therefore, it is not unusual that paramecia detect and are attracted to glutamate, which could very well indicate that bacteria, their food, are at hand. Mammalian taste has the added interesting aspect of synergism of glutamate with 5’ ribonucleotides, known as ‘umami’ taste ( Ugawa and Kurihara, 1994 Faurion, 1991). Lobsters ( Carr and Derby, 1986 Fine-Levy et al., 1987), fish ( Caprio et al., 1993), and mammals including humans ( Yamaguchi, 1987) can detect, that is, taste and/or smell, glutamate among other amino acids. Perhaps not as well appreciated is that glutamate also serves as an important environmental cue for many different organisms. Glutamate has been recognized as an important intercellular signal molecule for neurotransmission ( Tanabe et al., 1992 Wo and Oswald, 1995 Nakanishi, 1992). These studies are the first demonstration of a possible role for cyclic nucleotide second messengers in an attractant chemosensory transduction pathway in Paramecium. Results of behavioral tests of cells treated with protein kinase inhibitors also suggest that cyclic AMP is part of the signal transduction pathway for glutamate, but not for other attractant stimuli. An antagonist of glutamate, IMP, depolarizes the cells and decreases intracellular cyclic AMP by approx. Cyclic GMP does not change relative to basal levels over rapid or slower time courses of glutamate stimulation. We found that, in cells stimulated with glutamate, intracellular cyclic AMP increases by 30 mseconds and peaks at about sevenfold over basal levels by 200 mseconds. In order to determine whether the changes in cyclic AMP could be rapid enough to participate in stimulation as compared to slower processes such as adaptation, rapid kinetic measurements of cyclic AMP were made on whole cells by quenched-flow. Interestingly, other attractant stimuli, such as acetate and NH 4Cl, that similarly hyperpolarize the cell do not induce an increase in cyclic AMP observable at 30 seconds. We show here that by 1-30 seconds of stimulation, glutamate increases intracellular cAMP. It causes a hyperpolarization of the cell and smooth, relatively fast swimming that is characteristic of hyperpolarizing stimuli. Glutamate is an attractant stimulus to Paramecium tetraurelia.
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