Journal of Comparative Physiology B

https://doi.org/10.1007/s00360-019-01227-7

ORIGINAL PAPER

Ocean acidification: synergistic inhibitory effects of protons and heavy

metals on 45Ca uptake by lobster branchiostegite membrane vesicles
Dalen An1 · Aida Husovic1 · Laeequa Ali1 · Elizabeth Weddle1 · Lilian Nagle1 · Gregory A. Ahearn1,2

Received: 8 May 2019 / Revised: 11 June 2019 / Accepted: 1 July 2019

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Previous work with isolated outer membrane vesicles of lobster branchiostegite epithelial cells has shown that 45Ca2+ uptake
by these structures is significantly (p < 0.02) reduced by an incremental decrease in saline pH (increased proton concentra-
tion) and that this decrease is due to competitive inhibition between carrier-mediated transport of 45Ca2+ and hydrogen ions.
The present paper extends these previous findings and describes the combined effects of pH and cationic heavy metals on
branchiostegite uptake of 45Ca2+. Partially purified membrane vesicles of branchiostegite cells were produced by a homog-
enization/centrifugation method and were loaded with mannitol at pH 7.0. The time course of 1 mM 45Ca2+ uptake in a man-
nitol medium at pH 8.5 containing 100 µM verapamil (­ Ca2+ channel blocker) was hyperbolic and approached equilibrium at
30 min. This uptake was either significantly reduced (p < 0.05) by the addition of 5 µM Z ­ n2+ or essentially abolished with
the addition of 5 µM ­Cu . Increasing zinc concentrations (5–500 µM) reduced 1 mM Ca2+ uptake at pH 8.5 or 7.5 in a
2+ 45

hyperbolic fashion with the remaining non-inhibited uptake due to apparent non-specific binding. Uptake of 1 mM 45Ca2+ at
pH 8.5, 7.5, 7.5 + Zn2+, and 7.5 + Zn2+ + Cu2+ + Cd2+ in the presence of 100 µM verapamil displayed a stepwise reduction of
45
Ca2+ uptake with the addition of each treatment until only non-specific isotope binding occurred with all cation inhibitors.
45
Ca2+ influxes (15 s uptakes; 0.25–5.0 mM calcium + 100 µM verapamil) in the presence and absence of 10 µM Z ­ n2+ were
2+
both hyperbolic functions of calcium concentration. The curve with ­Zn displayed a transport Km twice that of the control
(p < 0.05), while inhibitor and control curve Jmax values were not significantly different (p > 0.05), suggesting competitive
inhibition between 45Ca2+ and Z ­ n2+ influxes. Analysis of the relative inhibitory effects of increased proton or heavy metal
45 2+
interaction with Ca uptake suggests that divalent metals may reduce the calcium transport about twice as much as a drop
in pH, but together, they appear to abolish carrier-mediated transport.

Keywords  Ocean acidification · Climate change · Homarus americanus · Lobster · Calcium transport · Gills ·
Branchiostegites · Pollution · Divalent cations, zinc, copper, cadmium · Competitive inhibition · Membrane vesicles ·
Electroneutral ­Ca2+/2H+ exchange · Antiport · Ion channel · Verapamil

Introduction alterations are affecting important biological processes of

organisms that live in these changing environments. Indus-
In recent years, the world’s oceans have been undergoing trial chemical pollutants, such as heavy metals including
significant changes in water chemistry that, at least in part, zinc, cadmium, and copper, enter the seas from a variety of
are a direct result of anthropocentric involvement. It is manufacturing activities, and have been shown to be toxic
increasingly apparent that many of these oceanic chemical to vertebrates and invertebrates alike (Ansari et al. 2004;
Tan et al. 2016; Baltas et al. 2017). Some of this toxicity
is a result of metal ions structurally resembling non-toxic
Communicated by H.V. Carey.
substances that are needed in specific amounts by organis-
* Gregory A. Ahearn mic essential transport and biosynthetic pathways, but which
gahearn2012@gmail.com cannot be used because of toxic ion interference (Flik et al.
1 1995).
Department of Biology, University of North Florida,
Jacksonville, FL 32224, USA While metal and organic pollutants have been affect-
2 ing water chemistry around the world for over a century,
Present Address: Jacksonville, USA

13
Vol.:(0123456789)

more recently increases in water temperature and hydrogen
ion concentration resulting from climate change and ocean
acidification have also had profound effects on marine biota
(Pörtner 2008; Brierley and Kingsford 2009; Whiteley
2011). In only a few years, the combination of increased
seawater temperature and acidity has markedly reduced shell
calcification processes of representatives of many inverte-
brate phyla and enhanced water temperature alone has led
to wide-spread coral bleaching and massive die-offs in the
Tropical Pacific and Caribbean parts of the world (Honisch
et al. 2012; Mostofa et al. 2016).
Recently, using American lobster (Homarus americanus)
apical gill membrane vesicles, we showed that incremental
decreases in incubation medium pH from pH 8.5–7.5 signifi-
cantly lowered the transport of 45Ca2+ into these vesicles and
that this effect was due to competitive inhibition between
45
Ca2+ and H­ + ions competing for a shared membrane carrier
protein (Nagle et al. 2018). The present study extends these
results by examining the effects of ­Zn2+, ­Cu2+, and ­Cd2+ on
45
Ca2+ uptake by these vesicles at pH 8.5 and 7.5, and sug-
gest that both protons and divalent metallic ions compete
with 45Ca2+ for uptake into lobster gill cells from seawater
in a synergistic manner, potentially reducing the available
hemolymph calcium available for calcium-dependent physi-
ological processes such as muscle contraction, nerve synap-
tic transmission, and exoskeletal calcification.
Materials and methods
Live Atlantic lobsters (Homarus americanus, 0.75 kg each)
were purchased from a commercial dealer in Jacksonville,
Florida and maintained unfed at 14 °C for up to 5 days in
artificial seawater (Instant Ocean) with a salt mixture kept at
34‰ salinity and pH 8.0. All animals used in this study were
either in intermolt or early premolt as assessed by the molt
classification scheme introduced by Aiken (1973). Although
apparent seasonal differences in lobster transport rates have
been observed over the course of a year (Piersol and Ahearn
2007), such seasonal variability was reduced by performing
all identical experiments within the same calendar month.
As described previously (Nagle et al. 2018), calcium
transport was followed in brachiostegite epithelial plasma
membrane vesicles from Atlantic lobsters by employing a
homogenization/centrifugation technique reported by Lucu
et al. (2008) applied to gill tissues of the shore crab Car-
cinus aestuarii. This method was similar to that reported
by Flik and Haond (2000) which produced a crude plasma
membrane fraction from gills, epipodites, and branchi-
ostegites of the European lobster, Homarus gammarus. In
both the previous investigations, crude plasma membrane
preparations consisting of both brush border and basolateral
plasma membranes were produced, but experiments were
13
Journal of Comparative Physiology B
conducted with these membranes using buffer compositions
that allowed for the distinction of basolateral transport prop-
erties while minimizing contributions from brush border
contaminants. In the present investigation, a similar crude
plasma membrane preparation was produced from Atlantic
lobster branchiostegite epithelial cells, but all experimental
solutions were only composed of mannitol and Hepes/Tris
buffers containing no sodium or ATP, thereby minimizing
calcium transport activities from basolateral components of
the mixture.
Partially purified membrane vesicles (PPMV) of lobster
branchiostegite epithelial cells were produced by adding
the scrapings from both gill chambers to ice-cold hypotonic
Buffer 1 (300 mM mannitol, 1 mM EGTA, 125 µM PMSF,
in 125 ml filtered water at pH 7.0) and homogenizing in a
blender for 3 min. Following homogenization, the prepara-
tion was centrifuged at 3000 rpm for 20 min in a Beck-
man Avanti centrifuge fitted with a JA25.5 rotor at 4 °C.
The resulting pellet was discarded and the supernatant was
centrifuged for 30 min at 20,000 rpm with the same rotor
at 4 °C. The resulting pellets were resuspended in 35 ml of
Buffer 2 (300 mM mannitol, 12 mM Hepes/Tris, in 500 ml
filtered water at pH 7.0) followed by ten strokes with a Pot-
ter–Elvehjem tissue homogenizer and a power drill. The
homogenized solution was again centrifuged at 20,000 rpm
for 30 min at 4 °C. The supernatant was discarded and the
pellet resuspended in 600 µl of Buffer 2. Vesiculation of the
final pellet was conducted using a 22-gauge needle to mix
the vesicles 20 times before use in a radioactive experiment.
Transport experiments using branchiostegite PPMV
were conducted at 23 °C using the Millipore filtration tech-
nique developed by Hopfer et al. (1973). Both long-term
and short-term incubations of membrane vesicles with 45Ca
(PerkinElmer, CT), using radioisotopic and liquid scintil-
lation methods, were previously described (Ahearn and
Franco 1990, 1993; Zhuang and Ahearn 1996). In most
cases, experiments were conducted using the calcium chan-
nel blocker, verapamil (100 µM), in the outside incubation
media, so that the effects of inhibitors on carrier-mediated
45
Ca transport could be assessed. 45Ca uptake values are
expressed as nmol (using specific activity of the isotope in
the medium) per milligram protein (Bio-Rad protein assay)
per filter. Within an experiment, three-to-four replicate ani-
mals were used and data are presented as means ± 1SEM.
Sigma Plot 10.0 (Systat Software, Inc., San Jose, CA) was
used to create time course and kinetics graphs. Statistical
differences of 45Ca uptake were determined using one-
way ANOVAs or Student’s t tests. Significant differences
between treatments were established at p < 0.05.
Valinomycin, amiloride, verapamil, Hepes, Tris, EGTA,
PMSF, and other reagent grade chemicals were obtained
from Fisher Scientific Co. (Waltham, MA) and Sigma
Chemical, Co. (St. Louis, MO).
Journal of Comparative Physiology B

Results

Effects of zinc and copper on the time course

of 1 mM 45Ca uptake

Partially purified membrane vesicles (PPMV) of lobster

branchiostegite epithelial plasma membranes were loaded
with 300 mM mannitol, 12 mM Hepes/Tris, at pH 7.0 and
incubated for 2.5, 5, 10, and 30 min in similar external media
at pH 8.5 containing 100 µM verapamil, 1 mM 45Ca2+ and
either 5 µM Z ­ nCl2 or 5 µM C ­ uCl2. Results in Fig. 1 show
that 45Ca2+ uptake in all external media was hyperbolic func-
tions of time approaching equilibrium by 30 min. 45Ca2+
uptake in the absence of either metallic ion most rapidly
reached apparent equilibrium which occurred around 30 min
of incubation at approximately 5 nmoles/mg protein. The Fig. 2  Effects of increasing zinc concentration on the 15  s influx of
addition of either ­Zn2+ or ­Cu2+ to the incubation medium 1 mM 45Ca2+ by lobster branchiostegite membrane vesicles at exter-
nal pH of 8.5 and 7.5 (intravesicular pH 7.0). All treatments included
led to significant reductions in the rate and apparent equilib- 100 µM verapamil. n = 4 animals. Symbols are means ± 1 SEM
rium of 45Ca2+ uptake with copper being the most inhibitory.
Because verapamil was added to all incubation media in this
experiment, all rates of uptake and their apparent equilibria and 3.94 ± 0.38 (pH 7.5) nmol/mg protein × 15 s (p < 0.05),
are assumed to be due to carrier transport only. suggesting that the increased proton concentration alone
at the lower pH significantly inhibited 1 mM 45Ca2+ influx
Effects of increasing zinc concentration on 15 s as disclosed in a previous publication (Nagle et al. 2018).
influxes of 1 mM 45Ca at pH 8.5 and 7.5 The addition of Z­ n2+ to media at both pH 8.5 and 7.5 led
to hyperbolic decay curves exhibiting apparent asymptotes
­ n2+ con-
Figure 2 shows the effects of increasing external Z (2.62 ± 0.52, pH 8.5; 2.29 ± 0.05, pH 7.5  nmol/mg pro-
centration (0, 5, 25, 100, and 500 mM) on 15 s influxes tein × 15 s) that were not significantly different from one
of 1 mM 45Ca2+ into PPMV loaded with 300 mM manni- another (p > 0.05). Because 100 µM verapamil and 500 µM
tol, 12 mM Hepes/Tris, at pH 7.0 and incubated in simi- ­Zn2+ were both present at each asymptotic value, these resid-
lar media containing 100 µM verapamil at pH 8.5 or 7.5. ual uptake values were considered non-specific isotope bind-
45
Ca2+ influxes at zero mM Z
­ n2+ were 6.19 ± 1.01 (pH 8.5) ing to vesicle membranes.

Effects of pH, verapamil, and metals on 15 s influxes

7
of 1 mM 45Ca2+
pH 8.5 + V
45Ca uptake (nmol/mg protein)

6 pH 8.5 + V + Zn Figure 3 shows the combined effects of proton concentra-

pH 8.5 + V + Cu
tion, verapamil, and heavy metals, added sequentially to the
5
incubation medium, on 15 s influx of 1 mM 45Ca2+ into
4 branchiostegite PPMV. Columns 1 and 3 compare 1 mM
45
Ca2+ influx at pH 8.5 and 7.5, indicating that a significant
3 (p < 0.05) decrease in calcium uptake as proton concentra-
tion was elevated. In both pH conditions, the addition of
2
100 µM verapamil significantly (p < 0.02) reduced uptake
1 of 45Ca2+ in the presence of the channel blocker (columns 2
and 4). A further significant (p < 0.02) drop in 45Ca2+ entry
0 occurred when 5 µM ­Zn2+ was added to the combination
0 10 20 30
of pH 7.5 and 100 µM verapamil (column 5). Finally, the
Time (minutes)
addition of 5 µM C ­ u2+ and 25 µM C ­ d2+ to media at pH
7.5 containing 100 µM verapamil and 5 µM Z ­ n2+ dropped
Fig. 1  Effects of zinc ­(Zn2+) and copper ­(Cu2+) on the time course uptake to an apparent inhibitory plateau that illustrated
of 1 mM 45Ca2+ uptake by lobster branchiostegite membrane vesicles
at external pH 8.5 (intravesicular pH 7.0). V = 100  µM verapamil; transport values (2.69 ± 2.53 ± 0.49 and 2.42 ± 0.38, nmol/
Zn = 5 µM; Cu = 5 µM. n = 3 animals. Symbols are means ± 1 SEM mg protein × 15 s, respectively) that were not significantly

13

45Ca influx(nmol/mg protein x 15 sec) 10
n = 3
8 3) pH 7.5
6
0
1 2 3 4
Treatments
Fig. 3  Effects of external pH, 100  µM verapamil, and metals on
the 15  s influx of 1  mM 45Ca2+ by lobster branchiostegite mem-
brane vesicles (intravesicular pH 7.0). ­Zn2+ = 5  µM; ­Cu2+ = 5  µM;
­Cd2+ = 25 µM. n = 3 animals. Symbols are means ± 1 SEM
different (p > 0.05) than those described previously at high
zinc concentrations (Fig. 2). This correspondence of inhibi-
tory concentrations of high metal concentrations on 45Ca2+
activity associated with PPMV in the two experiments sup-
ports the suggestion of residual uptake due to non-specific
isotope binding to membranes under these conditions.
Proportions of 1 mM 45Ca2+ influx inhibited
by protons (pH), metals, and channel blockers
Influx data displayed in Fig. 3 were used to determine what
proportions of total 1 mM 45Ca2+ influx in seawater at pH
8.5 were sequentially inhibited by an increase in proton con-
centration (decrease in pH), an addition of 100 µM vera-
pamil, and an increase in cationic heavy metals to assess the
relative effects of these inhibitors on the uptake pathways for
calcium accumulation. This analysis is shown in Fig. 4. A
reduction of pH from 8.5 to 7.5 induced a 17.8% decrease in
1 mM 45Ca2+ influx (e.g., 8.27–6.80 nmol/mg protein × 15 s),
while the addition of 100 µM verapamil to 45Ca2+ influx at
pH 8.5 blocked 35.3% of calcium transport (8.27–5.35 nmol/
mg protein × 15 s). Comparing 1 mM 45Ca2+ influx into vesi-
cles in media at pH 7.5 containing 100 µM verapamil (Fig. 3,
column 4) to vesicles in media at pH 7.5, 100 µM verapamil,
and the metals ­Zn2+, ­Cu2+, and ­Cd2+ (5 µM, 5 µM, 25 µM,
respectively; column 7, Fig. 3) showed that the combined
presence of the three metals reduced influx by 30.2% com-
pared to the reduction observed by pH and verapamil alone.
The residual 45Ca2+ radioactivity (non-specific binding) dis-
played in Fig. 3 as column 7 accounted for 29.3% of total
isotope activity at 1 mM 45Ca2+. These data can be used to
estimate the relative contribution of carrier-mediated and
channel uptake pathways by branchiostegite vesicles. At
13
Journal of Comparative Physiology B
1) pH 8.5
2) pH 8.5 + V
4) pH 7.5 + V
5) pH 7.5 + V + Zn
6) pH 7.5 + V + Zn + Cu
7) pH 7.5 + V + Zn + Cu + Cd
5 6 7
Fig. 4  Data from Fig.  3 illustrating the relative proportions of 15  s
influx of 1  mM 45Ca2+ inhibited by protons, metals, and channel
blockers. Residual activity refers to non-specific isotope binding to
vesicles after calcium carrier proteins and channels have been blocked
1 mM 45Ca2+ approximately 48% of calcium uptake occurred
by carrier transport (17.8% + 30.2%) which could be inhib-
ited by protons and metallic cations, while 35.3% took place
by way of a verapamil-inhibited calcium channel.
Heavy metals are competitive inhibitors of 45Ca2+
uptake by branchiostegite vesicles
Because Figs. 1, 2, and 3 show that the metallic cations
­Zn2+, ­Cu2+, and C­ d2+ reduced 45Ca2+ uptake by PPMV, an
effort was made to characterize the mechanism of this metal-
reducing effect. Figure 5 (upper curve) shows the effect of
increasing external 45Ca2+ concentration (0.25, 0.5, 1.0, 2.0,
and 5.0 mM) on 15 s influx of 45Ca2+ into vesicles contain-
ing 300 mM mannitol, 12 mM Hepes/Tris at pH 7.0 and
incubated in a similar external medium maintained at pH 8.5
and 100 µM verapamil. The lower curve was obtained under
similar conditions except that 10 µM Z
­ nCl2 was added to the
external media. The resulting data were curve-fit with Sigma
Plot 10.0 and describe single hyperbolic influx curves that
follow the Michaelis–Menten relationship:
( )
J = Jmax [Ca]∕ Km + [Ca] ,
Journal of Comparative Physiology B
Fig. 5  Effect of 10 µM zinc (­Zn2+) on 15 s hyperbolic influx of 45Ca
over a concentration range from 0.25 to 5 mM showing that zinc is a
competitive inhibitor of calcium uptake. Sigma Plot 10.0 was used to
curve-fit the resulting data as described in the text, see also kinetic
constant results in Table 1. All treatments included 100 µM verapamil
to block calcium channels. External pH = 8.5; intravesicular pH = 7.0.
n = 4. Symbols are means ± 1SEM
where J is 45Ca2+ influx in nmol/mg protein × 15 s, Jmax is
maximal influx, Km is an apparent binding affinity constant
(mM), and [Ca] is external calcium concentration. The
control curve displayed Km of 2.35 ± 0.59 mM and Jmax of
10.34 ± 1.35 nmol/mg protein × 15 s, while the data obtained
in the presence of ­Zn2+ had Km of 5.42 ± 0.21 and Jmax of
11.83 ± 0.28 nmol/mg protein × 15 s. The Km values were
significantly different (p < 0.05), while the Jmax values were
not significantly different (p > 0.05). Because of the differ-
ences between the Km figures and the similarity of the Jmax
figures, these graphic results suggest that external Z ­ n2+
acted as a competitive inhibitor of calcium influx into lobster
branchiostegite PPMV as discussed for competitive enzyme
kinetics by Segel (1975).
Discussion
During the last century, the world’s oceans and seas have
increasingly become repositories for anthropocentric waste
disposal, resulting in sediment and water column accumu-
lation of industrial chemical and solid byproducts such as
herbicides, insecticides, heavy metals, a wide array of water-
soluble and water-insoluble organic pollutants, and recently
micro-plastic and macro-plastic materials that remain in the
ecosystem for decades or longer (Ansari et al. 2004; Tan
et al. 2016; Baltas et al. 2017). In addition to these changes
in seawater composition, over the last 30–50 years, it has
become apparent that human-induced climate changes are
increasing the world’s water temperature by global warming
and increasing its acidity through excessive carbon dioxide
absorption from the atmosphere (Pörtner 2008; Brierley
and Kingsford 2009; Whiteley 2011; Honisch et al. 2012;
Mostofa et al. 2016). The effects of each of these perturba-
tions alone on vertebrate and invertebrate marine organisms
may be severe, potentially decreasing population sizes or
eliminating species all together. However, potential syner-
gistic negative interactions of more than one of these fac-
tors acting simultaneously on an animal population may be
catastrophic and lead to extinction at a faster rate than might
occur if only one factor were present (Whiteley 2011; Cebal-
los et al. 2017). The current study compares the inhibitory
effects of water acidification and heavy metal contamina-
tion as separate and combined factors affecting the uptake
of 45Ca by lobster gill membrane vesicles and suggests that
when present together, these two changes in water chemistry
may abolish carrier-mediated uptake of this important cation
from seawater potentially affecting the survival of individual
animals or their populations.
Epithelial regulation of calcium uptake
from the environment
Calcium is an essential ion needed for a wide variety of
structural (skeletal) and regulatory (nerve and muscle func-
tion; second messenger action, etc.) processes by both ver-
tebrate and invertebrate animals. Much of the physiologi-
cal calcium concentrations required by animals is obtained
through the diet and transferred to the blood across various
digestive tract epithelial cells. Many aquatic animals, such
as marine crustaceans and fishes, possess calcium transport
proteins in their gills for removing this ion from the envi-
ronment and contributing to its homeostatic maintenance
in the blood. Environmental factors affecting the uptake of
calcium from seawater and its subsequent transfer to the
blood across gill epithelial cells may lead to disturbances
in the homeostatic regulation of this ion and disruption of
physiological processes that depend on a consistent supply
of this essential factor.
Previous research with calcium transport regulation
by gastrointestinal (Ahearn 1996; Ahearn and Zhuang
1996; Zhuang and Ahearn 1996, 1998) and renal (Ahearn
and Franco 1990, 1993) organs of the American lobster,
Homarus americanus, and the freshwater crayfish, Procamb-
arus clarkii (Wheatly 1999; Wheatly et al. 1998, 2002; Gao
and Wheatly 2004), have described the carrier-mediated and
channel transport systems of hepatopancreatic and antennal
gland epithelial cells and have identified calcium/proton or
calcium/sodium antiporters, verapamil-inhibited calcium
channels, and calcium ATPases in these organs which simi-
larly regulate the uptake of calcium from the diet and its loss
in urine in animals from both environments. Protons, sodium
ions, and heavy metallic cations such as zinc, copper, and
13

cadmium were shown to share digestive and renal cal-
cium antiporters in these animals as a result of competitive
binding inhibition during the uptake process (Ahearn and
Franco 1990, 1993; Ahearn 1996; Ahearn and Zhuang 1996;
Zhuang and Ahearn 1996). As a result of this inhibition, less
dietary calcium was transferred into and across the digestive
tract or retained by the renal system of these animals.
Freire et al. (2008) in an extensive description of epithe-
lial structure and function of crustacean gill and excretory
epithelial cells proposed a theoretical model of calcium ion
transport proteins in previously understudied gill epithelial
cells adopting the antiporter, channel, and calcium-ATPase
paradigm proposed for digestive and excretory organs of
marine and freshwater crustaceans. This paradigm consisted
of an electrogenic 1­ Ca2+/1Na+ antiporter, an electroneutral
­1Ca2+/2Na+ antiporter, and a verapamil-inhibited C ­ a2+ chan-
nel on the outwardly facing (towards the seawater) epithelial
membrane and a ­Ca2+-ATPase and ­1Ca2+/3Na+ antiporter
on the basolateral (towards the blood) epithelial membrane.
Data presented previously (Nagle et al. 2018), and in the
current investigation, tested this theoretical model with par-
tially purified apical plasma membrane vesicles of lobster
branchiostegite epithelial cells. Nagle et al. (2018) examined
the effects of changes in saline pH on the uptake of 45Ca
and showed that vesicles displayed the highest accumula-
tion of calcium at pH 8.5 (near seawater pH) and the lowest
at pH values between 6.0 and 7.0. At pH 8.0, 45Ca uptake
was a biphasic function of external calcium concentration,
exhibiting both apparent diffusional and carrier-mediated
influx. At pH 8.5, influx was a hyperbolic function of cal-
cium concentration, exhibiting a Km value of 4.2 ± 0.3 mM
and J max of 9792 ± 439  pmol/mg protein × 15  s. How-
ever, at pH 7.5, influx Km was 8.3 ± 1.4 mM and Jmax was
10,732 ± 1250 pmol/mg protein × 15 s. Resulting Km values
were significantly different (p < 0.05), while Jmax values
were not (p > 0.05). Conclusions reached in this paper were
that branchiostegite apical plasma membrane 45Ca uptake
occurred by the combination of a verapamil-inhibited cal-
cium channel and an electroneutral calcium antiporter that
was competitively inhibited by protons. Results generally
supported the theoretical model presented by Freire et al.
(2008).
Synergistic inhibition of calcium uptake
by combined environmental agents
Results of the present study extend those previously dis-
cussed by Nagle et al. (2018), except in the present instance,
both pH and divalent heavy metal cations were varied inde-
pendently and in concert to assess their separate and com-
bined effects on 45Ca uptake by branchiostegite membrane
vesicles. All experiments in the present study were con-
ducted in the presence of 100 µM verapamil, which was
13
Journal of Comparative Physiology B
shown to block calcium channels in these membranes (Nagle
et al. 2018), so that effects of pH and metals on carrier-
mediated transport could be determined without diffusional
uptake through calcium channels. Figures 1 and 2 clearly
show that both 5 µM zinc and copper inhibit the uptake of
1 mM 45Ca at pH 8.5 with copper being a significantly more
potent inhibitor than zinc. However, with further elevation
of zinc concentration to 500 µM, total elimination of carrier-
mediated 45Ca uptake was observed. Residual 45Ca uptake at
500 µM zinc was assumed due to non-specific isotope bind-
ing to membranes. The concentrations of zinc and copper
used in these experiments are similar to those reported for
environmental water column and sediment metal concentra-
tions for various marine locations (Tan et al. 2016; Baltas
et al. 2017). Figure 3 illustrates the separate and combined
effects of increased acidity (change in pH from 8.5 to 7.5)
and increased metal ­(Zn2+, ­Cu2+, and ­Cd2+) concentrations
(5–25 µM) on vesicular influx of 1 mM 45Ca in the presence
of 100 µM verapamil. It is clear from this figure that each
of the separate environmental factors (i.e., pH and metal
concentration) partially inhibit carrier-mediated 45Ca uptake,
but that in combination (i.e., pH 7.5 + 5 µM or 25 µM met-
als), these factors completely abolish calcium uptake via
saturable transport systems leaving only non-specific iso-
tope binding, as was shown using 500 µM Z ­ n2+ in Fig. 2.
Inhibitory data from Fig. 3 were used in Fig. 4 to estimate
the relative effects of 100 µM verapamil, combined metal
concentrations, variable pH, and residual binding on 1 mM
45
Ca influx by lobster gill vesicles. The data suggest that
at pH 8.5, slightly less than half of 1 mM 45Ca transport
occurs through either channels or carriers with the remainder
due to non-transported, non-specific binding (residual activ-
ity). The nature of hydrogen ion and divalent metallic ion
inhibition of 45Ca influx is presented in Fig. 5 and Table 1.
In Fig. 5, 10 µM ­Zn2+ was used to inhibit 45Ca influx over
calcium concentrations ranging from 0.25 to 5.0  mM.
Both control (pH 8.5, no Z ­ n2+) and inhibited (+ 10 mM
2+
­Zn ) influx curves were hyperbolic functions of calcium
45
Table 1  Effects of pH and heavy metals on carrier-mediated Ca
influx kinetic constants
Treatment Km (mM) Jmax (nmol/mg Reference
protein × 15 s)
pH 8.5 (control) 4.20 ± 0.30 9.79 ± 0.43 Nagle et al. (2018)
pH 7.5 8.30 ± 1.40 10.73 ± 1.25 Nagle et al. (2018)
pH 8.5 (control) 2.35 ± 0.59 10.34 ± 1.35 This study
pH 8.5 + 10 µM 5.42 ± 0.21 11.83 ± 0.28 This study
zinc
All experiments conducted using 100  µM verapamil in incubation
media to block calcium channel 45Ca uptake. Three or four animals
were used in each experiment. Km values were significantly different
from one another (p < 0.05), while Jmax values were not (p > 0.05)
Journal of Comparative Physiology B
concentration following Michaelis–Menten kinetics, but the
inhibited influx curve was significantly (p < 0.05) lower than
the control curve at all calcium concentrations. Calculated
kinetic constants, Km (apparent binding affinities) and Jmax
(apparent maximal transport velocities), were obtained for
each curve using Sigma Plot 10.0 software curve-fitting
the data in Fig. 5 and the results are presented in Table 1.
Included in Table 1 are kinetic constants for proton-inhibited
45
Ca influx from Nagle et al. (2018) for comparison to the
values obtained with metal inhibition in the present study.
In both instances, inhibited Km values were significantly
(p < 0.05) higher (i.e., reduced binding affinity) than con-
trol values, while no significant differences (p > 0.05) were
observed between control and inhibited Jmax values. These
findings suggest that in both instances, proton inhibition or
metal inhibition, decreased 45Ca transport occurred by com-
petitive inhibition, where calcium and protons or calcium
and metal ions competed for binding to the same transport
carrier and that at high proton and metal concentrations,
little or no 45Ca transport was able to occur. While only two
treatment conditions were used to show competitive inhibi-
tion by protons (Nagle et al. 2018) or metals (Fig. 5), Segel
(1975) describes classic competitive enzyme kinetics with
the same graphic representation, providing support for the
nature of environmental agent inhibition proposed in Nagel
et al. (2018) and the present study.
Is a “universal” crustacean and fish gill calcium
regulatory system similarly affected by aquatic
acidification and metal pollution?
The data presented in this paper and in Nagle et al. (2018)
suggest that increased ocean acidification and pollution in
the coming decades may lead to potentially significant losses
of lobsters and other aquatic organisms that have similar gill
calcium transport systems which respond to these environ-
mental agents as discussed here. Considerable research has
been conducted on crab and fish gill structure and function
that suggest that part, or all, of the calcium transport para-
digm suggested by Freire et al. (2008), appear to occur in
these other groups of marine and freshwater animals. Whole
gill preparations and semi-purified gill basolateral plasma
membrane vesicles from crab (Carcinus maenas) and cray-
fish (Procambarus clarkii) have confirmed the presence of
a basolateral calcium ATPase and C ­ a2+/Na+ antiporter that
together help facilitate transepithelial transport of calcium
and provide a locus, where metallic cations such as zinc,
copper, and cadmium appear to affect the movement of cal-
cium through the epithelial cells (Flik et al. 1994; Pedersen
and Bjerregaard 1995; Lucu and Flik 1999; Wheatly 1999;
Henry et al. 2012). Crab gill apical uptake of calcium and
cadmium occurred via a verapamil- and lanthanum-inhibited
channel (Lucu 1994; Lucu and Obsersnel 1996; Sa et al.
2010; Ortega et al. 2014), and a carrier-mediated, saturable,
influx process (Sa et al. 2010; Henry et al. 2012; Ortega
et al. 2014). Freshwater Daphnia magna gill epipodite apical
surfaces displayed a 2­ Na+/H+ antiporter (Ahearn and Clay
1989; Ahearn and Franco 1990; Ahearn et al. 2001) that
was competitively shared by sodium, calcium, and protons
[apparent ­1Ca2+/2H+ antiporter (Nagle et al. 2018)], such
that in the presence of 1 mM calcium, a decrease in pH
from pH 8 to pH 6 resulted in an approximate 50% decrease
of 22Na+ influx and a tenfold decrease from pH 8 to pH 4
(Glover and Wood 2005). The results of these studies, test-
ing the effects of metallic pollutants and water acidification
on gills of various crustacean species, provide strong sup-
port for the theoretical transport model suggested by Freire
et al. (2008) and aid us in understanding, where and how
these environmental agents may interfere with normal ion
regulation.
Calcium transport by fish gills has been extensively stud-
ied in freshwater rainbow trout (Salmo gairdneri, Oncorhyn-
chus mykiss) and freshwater- and seawater-adapted tilapia
(Oreochromis mossambicus) using a variety of in  vivo
methods involving perfused tissues or organs and in vitro
studies employing semi-purified basolateral gill membrane
vesicles. To date, semi-purified apical membrane vesicle
preparations of fish gill cells have not been developed and
used to investigate direct calcium transfer across this cell
border (Flik et al. 1995). Fish gills appear to structurally
follow much of the transport paradigm described above for
crustacean gills, where in vivo fish gill preparations pos-
sess an apical ion channel that allows the entry of calcium,
cadmium, and zinc followed by basolateral exit from the
gill cells via a ­Ca2+-ATPase or a ­Ca2+/Na+ antiporter (Flik
et al. 1995). In vivo influx studies of 45Ca uptake suggest
that both zinc and cadmium are competitive inhibitors of this
uptake process, but the site of cation interaction is unclear
as to whether it occurs on the apical membrane via a shared
uptake carrier process, that has not yet been identified, or
at the basolateral ­Ca2+-ATPase prior to efflux from the cell
(Verbost et al. 1987, 1988, 1989, 1994; Spry and Wood
1989; Hogstrand et al. 1994, 1996). In all cases, the interac-
tions between calcium and either zinc or cadmium resulted
in reduced calcium transfer across the gill epithelium.
Effects of aquatic acidification on fish gill calcium
transport measured with in vivo preparations have gener-
ally showed inhibitory interactions between the two cati-
ons resulting in complete blockage of calcium transport at
very low pH values (e.g., pH 4.0) (Hobe and Laurent 1984).
However, it has also been reported that the addition of
small amounts of calcium to acid trout farm water (e.g., pH
3.0–4.8) increases survival rates of fingerlings by partially
blocking acid exposure (McDonald et al. 1980). Because
there are no reported apical shared carrier proteins in fish
gill cell membranes accommodating protons, calcium, and
13

cationic metals as suggested for crustacean gills (Nagle et al.
2018), the nature of how protons and calcium ions interact at
fish gill cells is unclear, but is likely more complex than sim-
ple competitive inhibition, as considerable structural damage
occurs at low pH which affects the general ion transport
capacity of the gill cells (Bonga et al. 1990).
Summary
Synergistic inhibitory effects of water acidity and metallic
divalent cations were investigated using lobster branchioste-
gite gill apical plasma membrane vesicles. The metals, cop-
per, zinc, and cadmium inhibited 1 mM 45Ca uptake by these
vesicles, completely blocking carrier-mediated isotope accu-
mulation at maximal metal concentrations. These inhibitory
effects between calcium and metals occurred by competitive
binding to a shared apical carrier protein. A verapamil-inhib-
ited calcium channel was also present on these apical mem-
branes and could be blocked during transport experiments to
characterize the properties of carrier function. When added
sequentially, increased proton concentration and increased
metal concentration were able to completely block carrier-
mediated 45Ca influx in the absence of channel activity. The
results of this study, and those of a previous investigation
(Nagle et al. 2018) support a theoretical transport model for
crustacean gill epithelial cells as proposed by Freire et al.
(2008). This crustacean model is generally similar to the cal-
cium and metal transport system present in fish gills, except
that the presence of apical carrier-mediated calcium uptake
in fish has been difficult to isolate and may be absent.
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