We examined the effect of carvacrol on Ca2+cytin the model yeast Saccharomycescerevisiae expressing the protein aequorin, after reconstitution with itsprosthetic group coelenterazine. Ca2+-dependent luminescence of the aequorin-coelenterazinephotoprotein complex was monitored following addition of 0.6 mg/ml carvacrol quantified using aphotomultiplier tube. The treatments used in the experiment were wild-type, lanthanumchloride-treated cells and knockout mutant cells. Carvacrol induced a biphasic elevation in Ca2+cytin wild type cells as shown in Figure 2.

The first phase was rapid (66.20seconds) and of greater magnitude (0.51 µM) compared to the second phase (189.88 seconds, 0.23 µM)(Table 1).

The first peak was present in all three treatment conditions,however with similar reduced total Ca2+cyt elevations lanthanumchloride and mutant cells (335.08 and 359.87 µM). Magnitude alsowas decreased by approximately 67% for both lanthanum chloride and mutant,compared to wild-type as displayed in Figure 2. The transient second phase elevation following addition of carvacrol wascompletely abolished in mutant cells and those treated with lanthanum chloride.The timing of the first phase in mutant cells was considerably prolonged (99.

88seconds) compared to wild-type and lanthanum chloride (66.20 and 71.36 seconds)(Tables 1-3). This data displays that mutant and lanthanum chloride treatedcells are less sensitive to the carvacrol induced changes in cytosolic calciumconcentrations.  There were no statisticallysignificant differences between the three group Ca2+cyt as determined by one-way ANOVA (F(2,12) = 3.81, p = 0.052).

Table 4displays the ANOVA analysis table. An independent t-test concluded that there wereno significant difference between the mean total increase in Ca2+cytof lanthanum chloride and mutant, and wild type and mutant (p > 0.05). Thiscould be due to the large SEM in wild-type cells, which if reduced throughincreased precision of results could lead to statistical significance betweenwild-type and mutant.

There was a significant difference however between meantotal increase in Ca2+cyt of lanthanum chloride andwild-type (p < 0.05). We examined the effects of carvacrolon cytosolic calcium changes in different treatments (10Mm lanthanum chloride andan unknown mutant). These observations show that carvacrol induces Ca2+cyt elevationsin Saccharomyces cerevisiae, and mutant and lanthanum chloride treated cells areless sensitive to the effects, with reduced magnitude and lack of second phase.Both lanthanum chloride and the mutant have similar inhibitory effects on Ca2+channels in the plasma membrane because there is no significant differencebetween the two conditions in terms of magnitude and total Ca2+cyt  elevations. Similarly,Rao et al., (2010) foundthat phenolic compounds including carvacrol resulted in Ca2+cyt  elevations, disrupting calciumhomeostasis. Carvacrol has been found to act as an agonist/antagonist fortransient receptor channels (TRP) and also voltage gated calcium channels (Dantaset al.

, 2015).   The Ca2+ signature of a cellis shaped by Ca2+ influx from extracellular pools via theplasma membrane and intracellular stores result in Ca2+ into the cytosol fromdiscrete compartments within the cell (such as the vacuole in fungi), which contribute to stimulus induced Ca2+cyt increases.Ca2+ efflux transporters rapidly remove Ca2+ from the cytosol to restore Ca2+cyt toresting value (McAinsh and Pittman, 2009). In yeast, Ca2+cyt  is tightly regulated and maintained at50-200 nM (Cui et al., 2009). It has been found that eugenol (a phenoliccompound like carvacrol) induced Ca2+cyt increases have two componentsincluding a cch1p-dependent Ca2+ influx induced immediately after eugenoladdition and a cch1p-independent Ca2+ influx which is delayed and prolonged(Roberts, McAinsh and Widdicks, 2012).

Cch1p is a high-affinity,voltage-gated calcium influx channel located on S. cerevisiae plasma membrane. It has been found that deletion ofcch1p reduces Ca2+cyt in yeast (Loukin et al., 2008).  This isconsistent with the biphasic calcium changes found here. Lanthanumchloride used here is a non-selective plasma membrane Ca2+ channel blocker,therefore reducing levels of Ca2+cyt . Thedata shows that both the lanthanum chloride and mutant cells result in loweredCa2+ influx into the cytosol and magnitude compared to wild-type.

As the resultsfrom these two treatment conditions were similar, the knockout gene in themutant may also be a plasma membrane calcium channel like cch1.  The second phase has beenshown to derive from calcium influx from the vacuolar store via the Yvc1channel which is part of the transient receptor potential Ca2+ channel (TRP)family (Gupta et al., 2003;Palmer et al., 2001).

Both Cch1p and Yvc1 are depicted in Figure 3 showingcalcium influx into the cytosol from extracellular and intracellular stores. TheYvc1 channel is regulated by cytosolic calcium levels (Cui et al., 2009) therefore could be involved in a positive feedback calciummobilsing mechanism of calcium induced calcium release (CICR). Palmer et al., (2001) also stated that it was more likely thatYvc1 is a CICR channel due to the concentration of free vacuolar Ca2+at physiological pH.

This would therefore explain the abolished second phasefound in the mutant and lanthanum chloride treated cells (Figure 2). If themutant is devoid in cch1 and lanthanum chloride also targets this channel, thiswould explain the similar carvacrol induced Ca2+cyt changes. There would be less influx of Ca2+ from extracellularsources into the cytoplasm through cch1p, meaning lack of CICR from Yvc1 in thevacuole and subsequently no slower, lower magnitude second phase. As carvacrolis a known agonist of voltage gated calcium channels, this would explain whylanthanum chloride and the mutant treatments affect Ca2+cyt influx. 

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