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Biosci. Biotechnol. Biochem., 66 (5), 1069–1074, 2002

Phenylethylamine Induces an Increase in Cytosolic Ca 2+ in Yeast Reinhard PINONTOAN,1,* Svetlana KRYSTOFOVA,1 Tomonori KAWANO,2,** Izumi C. MORI,1,*** Frederick I. TSUJI,3 Hidetoshi IIDA,4,5 and Shoshi MUTO1,2,† 1Nagoya

University Bioscience Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan 3Marine Biology Research Division, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093, U.S.A. 4Department of Biology, Tokyo Gakugei University, Koganei, Tokyo 184-8501, Japan 5CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan 2Graduate

Received December 4, 2001; Accepted January 7, 2002

b-Phenylethylamine (PEA) induced an increase in cytosolic free calcium ion concentration ([Ca 2＋]c) in Saccharomyces cereviseae cells monitored with transgenic aequorin, a Ca 2＋-dependent photoprotein. The PEA-induced [Ca 2＋]c increase was dependent on the concentrations of PEA applied, and the Ca 2＋ mostly originated from an extracellular source. Preceding the Ca 2＋ in‰ux, H2 O2 was generated in the cells by the addition of PEA. Externally added H2 O2 also induced a [Ca 2＋]c increase. These results suggest that PEA induces the [Ca 2＋]c increase via H2 O2 generation. The PEA-induced [Ca 2＋]c increase occurred in the mid1 mutant with a slightly smaller peak than in the wild-type strain, indicating that Mid1, a stretchactivated nonselective cation channel, may not be mainly involved in the PEA-induced Ca 2＋ in‰ux. When PEA was applied, the MATa mid1 mutant was rescued from a-factor-induced death in a Ca 2＋-limited medium, suggesting that the PEA-induced [Ca 2＋]c increase can reinforce calcium signaling in the mating pheromone response pathway.

Key words:

calcium signaling; cytosolic Ca 2+; hydrogen peroxide; b-phenylethylamine; Saccharomyces cerevisiae

b-Phenylethylamine (PEA), an aromatic monoamine, is a secondary metabolite normally present at very low concentrations, and functions in neural systems in mammals.1) The oxidative deamination of

PEA generates H2 O2, and induces the release of Ca 2+ from isolated brain mitochondria.2) In plants, PEA and its derivatives are wide spread.3) PEA induces the generation of reactive oxygen species (ROS) and a [Ca 2+]c increase in tobacco suspension cultures and may trigger a signal transduction cascade leading to plant defense responses.4,5) A similar ROS generation and [Ca 2+]c increase were observed in a tobacco suspension culture treated with salicylic acid, a compound tightly involved in systemic acquired resistance in pathogen-attacked plants.6,7) Salicylic acid induces a [Ca 2+]c increase also in yeast cells.8) Various stimuli including a mating pheromone9,10) and osmotic stress11) have been shown to induce [Ca 2+]c increases in yeasts. The mating pheromone response pathway is known to be dependent on calcium signaling. A [Ca 2+]c increase due to Ca 2+-in‰ux from an extracellular source induced by mating pheromone occurs in the mating response pathway. This [Ca 2+]c increase is a crucial factor for promoting the survival of yeast cells exposed to mating pheromone in low Ca 2+ media,9,10) and the in‰ux is facilitated by two plasma membrane calcium permeable channels, Mid110,12) and Cch1.12,13) The yeast mating pheromone response pathway has been used for studying proteins whose activation is depending on Ca 2+.14,15) Therefore, the yeast mating pheromone response pathway may serve as a good system to examine whether the increase of [Ca 2+]c may be involved in calcium signaling that causes any cellular

To whom correspondence should be addressed. * Present address: Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan ** Present address: Graduate School of Sciences, Hiroshima University, Kagamiyama 1-3-1, Higashihiroshima, Hiroshima 739-8526, Japan *** Present address: Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0116, U.S.A. Abbreviations : BAPTA,1,2-bis(2-aminophenoxy)-ethane-N,N,N?,N?-tetraacetic acid; [Ca 2+]c, cytosolic free Ca 2+ concentration; coelenterazine, 2-(p-hydroxybenzoyl)-6-(hydroxyphenyl)-3,7-dihydroimidazo [1,2-a]pyrazin-3-one; H 2 O2, hydrogen peroxide; PEA, bphenylethylamine; rlu, relative luminescence units; ROS, reactive oxygen species †

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responses. In this study, we report that PEA induces a [Ca 2+]c increase in yeast and the [Ca 2+]c increase is able to reinforce calcium signaling in the yeast mating pheromone response.

Materials and Methods Yeast strains and transformation of apoaequorin. Yeast strains used in this study were a standard laboratory wild-type yeast strain, W303-1A, with a genotype MAT a his3 leu2 trp1 ade2 ura3, and a mid1 mutant strain H301, MAT a his3-D1 leu2-3,112 trp1-289 ura 3–52 sst1-2 mid1 and its parental wildtype strain H207, MAT a his3-D1 leu2-3,112 trp1-289 ura3-52 sst1-2.10) A 2-mm yeast expression vector pKT11 containing a full-length cDNA encoding apoaeqorin under regulation of a glucose-inducible promoter, TDH3 was introduced into yeast cells by a yeast transformation method using lithium acetate. Cells were grown on a selective complete-dextrose medium without uracil (SD-Ura medium) at 309C for 3 d as pKT11 contains the URA3 gene as a selection marker. After aequorin was reconstituted as described below, the cell suspensions of Ura+ transformants were treated with the same volume of 2 M CaCl2 in 20z ethanol to test their ability to produce aequorin luminescence. Chemicals. Chemically synthesized 2-( p-hydroxybenzoyl)-6-(hydroxyphenyl)-3,7-dihydroimidazo [1,2-a]pyrazin-3-one (coelenterazine) was a generous gift from Prof. M. Isobe of Nagoya University. PEA and ethylamine were purchased from Wako Pure Chemical Industries (Osaka, Japan). Benzylamine and serotonin were from Nacalai Tesque Inc. (Kyoto, Japan) and 1,2-bis(2-aminophenoxy)ethane-N,N,N?,N?-tetraacetic acid (BAPTA), was from Dojindo Laboratories, Inc. (Kumamoto, Japan). 2?,7?-Dichloro‰uorescin diacetate was from Molecular Probes Inc. (Eugene, OR, U.S.A.). Other chemicals used were the highest grades commercially available. Monitoring of Ca 2+-dependent aequorin luminescence. For monitoring Ca 2+-dependent aequorin luminescence, mid log phase yeast cells grown on SDUra plates were incubated in SD medium without CaCl2 (SD-Ca medium) with 50 mM coelenterazine at 309C for 30 min to reconstitute aequorin from apoaequorin.16) Then the cells were centrifuged to remove the excess of coelenterazine, and suspended in SD medium containing 100 mM CaCl2 (SD.Ca100 medium). One hundred ml of yeast culture with a density of 3×108 cells W ml was taken in a plastic cuvette and Ca 2+-dependent aequorin luminescence was measured with a luminometer (Lumicounter 1000; Microtech-Nition Co., Funabashi, Japan) connected

Fig. 1. PEA-Induced [Ca 2+]c Increase in Wild-type Yeast Cells. A typical representative trace of aequorin-luminescence obtained from 5 replications at each PEA concentration. Arrows indicate the addition of PEA.

with a pen recorder as described previously.8,17) The luminescence was expressed in relative luminescence units (rlu).

Detection of H2 O2 generation in yeast cells. Stationary-phase yeast cells were incubated with dichloro‰uorescin diacetate (10 mg W ml), a membrane-permeable ‰uorescent probe for H2 O2, at 309 C for 1 h. The cells were collected by centrifugation and washed with and suspended in SD medium. Intracellular H2 O2 generation in yeast cells were measured by monitoring ‰uorescence of dichloro‰uorescein, the oxidized product of dichloro‰uorescin, which was a hydrolysis product of dichloro‰uorescin diacetate by intracellular esterases, at excitation and emission wavelengths at 488 and 524 nm, respectively.4) Observation of morphology and viability of cells. For microscopic observations, yeast cells were grown in SD.Ca100 medium at 309 C to reach a density of ml, and exposed to 6 mM a-factor, a mat2×106 cells W ing pheromone. At designated times, a portion of the culture was mixed with the equal volume of 0.01z methylene blue W 2z sodium citrate solution.9) The morphology and viability of the cells were examined under a diŠerential interference-contrast microscope.

Results PEA-induced [Ca 2+]c increase Figure 1 shows that the addition of PEA up to 2.5 mM induced a transient [Ca 2+]c increase in a dosedependent manner in the wild-type yeast cells. With 2.5 mM PEA, [Ca 2+]c reached a peak within a few seconds, then decreased to a sustained level slightly higher than the resting level, and this level lasted over the observing time (3 min). To identify the source of the increased [Ca 2+]c, the eŠect of BAPTA, a Ca 2+chelator, on the PEA-induced [Ca 2+]c increase was

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Fig. 2. Inhibitory EŠect of BAPTA on the PEA-Induced [Ca 2+]c Increase in Wild-type Yeast Cells. Aequorin-luminescence produced by PEA was deˆned as 100z. Bars represent SE of 5 replicates.

examined. As shown in Fig. 2, removal of extracellular Ca 2+ by incubating yeast culture with 0.2 mM BAPTA for 5 min before the application of 2.5 mM PEA reduced the peak height of the PEA-induced [Ca 2+]c increase by 87z in the wild-type yeast cells, suggesting that Ca 2+ was supplied from an extracellular source. However, a possible minor contribution of release from intracellular Ca 2+ store could not be ruled out since BAPTA did not completely suppress the stimulatory eŠect of PEA. Note that SD-Ca medium contains 0.2 mM Ca 2+,9) thus the yeast suspension is essentially Ca 2+-free in the presence of 0.2 mM BAPTA.

Possible involvement of H2 O2 in the PEA-induced [Ca 2+]c increase In mammals, PEA is a substrate of amine oxidases. The oxidative deamination of PEA by amine oxidases generates phenylethylaldehyde, ammonia, and H2 O2. To understand a possible mechanism of the PEA induced [Ca 2+]c increase in yeast, the generation of intracellular H2 O2 was monitored by using dichloro‰uorescin, which is a non‰uorescent compound and is converted to ‰uorescent dichloro‰uorescein by H2 O2-dependent oxidation. When 10 mM PEA was added to yeast culture, a rapid increase in ‰uorescence of dichloro‰uorescein followed by a gradual increase was observed (Fig. 3A). The addition of 50 mM H2 O2 increased the intensity of ‰uorescence, conˆrming that a H2 O2dependent ‰uorescence was generated in yeast cells. These results also indicate that PEA permeated into the cells and was oxidized to generate H2 O2.

Fig. 3. Involvement of H2 O2 in a [Ca 2+]c Increase in Wild-type Yeast Cells. A, PEA-induced intracellular H2 O2 generation (‰uorescence of dichloro‰uorescein). B, [Ca 2+]c increase induced by H 2 O2 or PEA. Arrows indicate addition either PEA or H2 O2.

Next, the eŠects of H2 O2 administration on [Ca 2+]c were investigated in yeast cells expressing aequorin. Though exogenous H2 O2 induced a [Ca 2+]c increase, the amplitude of the peak produced by 10 mM H2 O2 was much lower than that by 2.5 mM PEA (Fig. 3B). Although H2 O2 could easily permeate the cell membrane, the intracellularly generated H2 O2 seemed more eŠective on the [Ca 2+]c increase.

EŠects of several monoamines on a [Ca 2+]c increase Potencies of several monoamines such as benzylamine, serotonin, and ethylamine in inducing a [Ca 2+]c increase were compared to PEA at a dose of 2.5 mM. As shown in Fig. 4, benzylamine, which has a structure similar to PEA, could induce a [Ca 2+]c increase in wild-type yeast cells. However, the amplitude of the peak induced by benzylamine was much

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Fig. 4. Potencies of Monoamines in Inducing a [Ca 2+]c Increase in Wild-type Yeast Cells. Aequorin-luminescence produced by PEA was deˆned as 100z. BA, benzylamine; EA, ethylamine; SER, serotonin. Bars represent SE of 4–5 replicates.

lower (23z) than that induced by PEA. Neither serotonin, an aromatic monoamine, nor ethylamine, an aliphatic monoamine, induced a [Ca 2+]c increase.

PEA-induced [Ca 2+]c increase involved in the mating pheromone response pathway To ˆnd whether the Mid1 channel is involved in the PEA-induced Ca 2+ in‰ux, the eŠect of PEA on the [Ca 2+]c increase was done in the mid1 mutant (strain H301). The addition of PEA up to 2.5 mM induced a [Ca 2+]c increase in the mid1 mutant, but the peak was smaller in the mutant than either in its parental strain H207 (data not shown) or a wild-type strain (Fig. 1, Fig. 5B, C). This result suggests that the Mid1 channel is involved in the PEA-induced Ca 2+ in‰ux in part and that there is another Ca 2+-permeable channel responsible for more of it. Since PEA was able to induce a [Ca 2+]c increase in the mid1 mutant, we tested whether this increase rescues the mutant from the mating pheromone-induced death, a phenotype of the mutant. It has been shown that the mid1 mutant cells die after diŠerentiation into shmoos when incubated in a low Ca 2+ medium, SD.Ca100.10) Cells of the mid1 mutant were incubated for 8 h with 6 mM a-factor in the presence and absence of PEA, and cell viability and morphology were then examined. As shown in Fig. 5D–F and Table 1, the mid1 cells not treated with PEA lost viability (16z viable), but those treated with 0.5 and 2.5 mM PEA maintained viability as high as 32 and 76z, respectively. In the same experimental conditions, the viability of its parental strain (H207) was more than 90z (Table 1). Cell morphology was also recovered by PEA (Fig. 5E, F). Incubation with a-factor in the absence of PEA led the mid1 cells to diŠerentiate into shmoos

Fig. 5. PEA-Induced [Ca 2+]c Increase and Morphology of the mid1 Mutant Exposed to a-Factor in the Presence of Various Concentrations of PEA. Representative traces of PEA-induced aequorin-luminescence obtained from each 5 replications (A–C). Arrows indicate addition of PEA. Morphology of cells (D–F). Cells were treated with the indicated concentrations of PEA, exposed to 6 mM a-factor for 8 h, and stained with methylene blue. Dark cells (shmoos) are dead cells and white cells (shmoos) are living cells.

having a single mating projection and then to die (Fig. 5D). PEA made the mid1 mutant to diŠerentiate into viable shmoos having two projections, a phenotype of the parental strain (Fig. 5E, F). These results suggest that the PEA-mediated Ca 2+ in‰ux system can compensate for loss of the function of the Mid1 channel in the mating pheromone response.

Discussion In eukaryotic cells, [Ca 2+]c is maintained at a low

Phenylethylamine-Induced Ca 2+ Increase in Yeast Table 1. Viability of the mid1 Mutant Exposed to 6 mM a-Factor for 8 h in the Presence of PEA Viability (z) PEA (m M)

Parental strain (H207)

mid1 mutant (H301)

0 0.5 2.5

93.2 (543) 95.2 (583) 94.3 (618)

16.1 (545) 31.9 (527) 75.7 (540)

Numbers in brackets indicate total numbers of counted cells.

basal level of around 100 nM, and there is accumulating evidence that supports changes in [Ca 2+]c are common signaling events. Plant cells respond to a variety of biotic and abiotic stimuli by increasing [Ca 2+]c. PEA, a secondary metabolite that may be involved in the plant defense response, induces a [Ca 2+]c increase in a tobacco suspension culture as a consequence of ROS generation.4,5) As much as 75z of ROS generation in the apoplast is catalyzed by peroxidase and copper amine oxidase. The remaining part of ROS is generated intracellularly by peroxidase-like and monoamine oxidase-like activities. In mammals, the oxidative deamination of aromatic monoamines including PEA generates H2 O2.2,18) This reaction is catalyzed by ‰avin-containing monoamine oxidases. Mammals have two types of monoamine oxidases, A and B. They are distinguished from each other by their substrate preference and susceptibility to inhibitors. Monoamine oxidase A prefers serotonine and dopamine as its substrates, while monoamine oxidase B does PEA. The PEA-induced Ca 2+ release from isolated rat-brain mitochondria is due to H2 O2 generation catalyzed by monoamine oxidase B.2) In this study, because PEA and benzylamine, but not serotonin and ethylamine, induced [Ca 2+]c increases in yeasts, we speculate that a monoamine oxidase B-like enzyme generated H2 O2. In yeast, FMS1 encoding a potential putative ‰avin-containing monoamine oxidase,19) has been characterized, and recently demonstrated to play a role in the metabolism of pantothenic acid,20) however, its monoamine oxidase activity remains to be elucidated. There has been a body of evidence showing that H2 O2 induces a [Ca 2+]c increase in eukaryotic cells21–24) as well as in prokaryotic cells.25) Thus, we proposed that PEA permeates into yeast cells, generates intracellular H2 O2, and subsequently induces a [Ca 2+]c increase. The in‰ux of extracellular Ca 2+ is supposed to be facilitated by Ca 2+ channels in the plasma membrane. MID1 has been demonstrated to be a gene encoding a plasma membrane protein required for Ca 2+ in‰ux and mating response in S. cerevisiae.10) Recently, Mid1 was identiˆed as a stretch-activated nonselective cation channel permeable to Ca 2+.26) Our study showed that Mid1 might not be a major

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PEA-induced Ca 2+ in‰ux channel. Therefore, an unidentiˆed Ca 2+-permeable channel(s) with sensitivity to H2 O2 may facilitate the PEA-induced Ca 2+ in‰ux across the plasma membrane of yeast.

Acknowledgments The authors acknowledge Prof. M. Isobe's gift of coelenterazine. R. P. and S. K. acknowledge the Japan Society for Promotion of Science for postdoctoral fellowships for foreign researchers. This work was supported by a Grant-in-Aid for Scientiˆc Research on Priority Areas (09274212, 10170216) from the Ministry of Education, Science, Sports, and Technology, Japan to S.M.

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