We examined the effect of carvacrol on Ca2+cyt
in the model yeast Saccharomyces
cerevisiae expressing the protein aequorin, after reconstitution with its
prosthetic group coelenterazine. Ca2+-dependent luminescence of the aequorin-coelenterazine
photoprotein complex was monitored following addition of 0.6 mg/ml carvacrol quantified using a
photomultiplier tube. The treatments used in the experiment were wild-type, lanthanum
chloride-treated cells and knockout mutant cells.


Carvacrol induced a biphasic elevation in Ca2+cyt
in wild type cells as shown in Figure 2. The first phase was rapid (66.20
seconds) 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 lanthanum
chloride and mutant cells (335.08 and 359.87 µM). Magnitude also
was 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 was
completely abolished in mutant cells and those treated with lanthanum chloride.

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The timing of the first phase in mutant cells was considerably prolonged (99.88
seconds) compared to wild-type and lanthanum chloride (66.20 and 71.36 seconds)
(Tables 1-3). This data displays that mutant and lanthanum chloride treated
cells are less sensitive to the carvacrol induced changes in cytosolic calcium


There were no statistically
significant differences between the three group Ca2+cyt as determined by one-way ANOVA (F(2,12) = 3.81, p = 0.052). Table 4
displays the ANOVA analysis table. An independent t-test concluded that there were
no significant difference between the mean total increase in Ca2+cyt
of lanthanum chloride and mutant, and wild type and mutant (p > 0.05). This
could be due to the large SEM in wild-type cells, which if reduced through
increased precision of results could lead to statistical significance between
wild-type and mutant. There was a significant difference however between mean
total increase in Ca2+cyt of lanthanum chloride and
wild-type (p < 0.05). We examined the effects of carvacrol on cytosolic calcium changes in different treatments (10Mm lanthanum chloride and an unknown mutant). These observations show that carvacrol induces Ca2+cyt elevations in Saccharomyces cerevisiae, and mutant and lanthanum chloride treated cells are less 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 difference
between the two conditions in terms of magnitude and total Ca2+cyt  elevations. Similarly,
Rao et al., (2010) found
that phenolic compounds including carvacrol resulted in Ca2+cyt  elevations, disrupting calcium
homeostasis. Carvacrol has been found to act as an agonist/antagonist for
transient receptor channels (TRP) and also voltage gated calcium channels (Dantas
et al., 2015).  


The Ca2+ signature of a cell
is shaped by Ca2+ influx from extracellular pools via the
plasma membrane and intracellular stores result in Ca2+ into the cytosol from
discrete 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 to
resting value (McAinsh and Pittman, 2009). In yeast, Ca2+cyt  is tightly regulated and maintained at
50-200 nM (Cui et al., 2009). It has been found that eugenol (a phenolic
compound like carvacrol) induced Ca2+cyt increases have two components
including a cch1p-dependent Ca2+ influx induced immediately after eugenol
addition 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 of
cch1p reduces Ca2+cyt in yeast (Loukin et al., 2008).  This is
consistent with the biphasic calcium changes found here. Lanthanum
chloride used here is a non-selective plasma membrane Ca2+ channel blocker,
therefore reducing levels of Ca2+cyt . The
data shows that both the lanthanum chloride and mutant cells result in lowered
Ca2+ influx into the cytosol and magnitude compared to wild-type. As the results
from these two treatment conditions were similar, the knockout gene in the
mutant may also be a plasma membrane calcium channel like cch1.


The second phase has been
shown to derive from calcium influx from the vacuolar store via the Yvc1
channel 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 showing
calcium influx into the cytosol from extracellular and intracellular stores. The
Yvc1 channel is regulated by cytosolic calcium levels (Cui et al., 2009) therefore could be involved in a positive feedback calcium
mobilsing mechanism of calcium induced calcium release (CICR). Palmer et al., (2001) also stated that it was more likely that
Yvc1 is a CICR channel due to the concentration of free vacuolar Ca2+
at physiological pH. This would therefore explain the abolished second phase
found in the mutant and lanthanum chloride treated cells (Figure 2). If the
mutant is devoid in cch1 and lanthanum chloride also targets this channel, this
would explain the similar carvacrol induced Ca2+cyt changes. There would be less influx of Ca2+ from extracellular
sources into the cytoplasm through cch1p, meaning lack of CICR from Yvc1 in the
vacuole and subsequently no slower, lower magnitude second phase. As carvacrol
is a known agonist of voltage gated calcium channels, this would explain why
lanthanum chloride and the mutant treatments affect Ca2+cyt influx. 


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