Articles in PresS. Am J Physiol Regul Integr Comp Physiol (June 7, 2017). doi:10.1152/ajpregu.00208.2017
1
2
3
4
5
Heads you gain, tails you lose:
6
Editorial focus on “Flexible ammonia handling strategies using both cutaneous and
7
branchial epithelia in the highly ammonia tolerant Pacific hagfish”
8
KM Gilmour
9
10
11
Department of Biology, University of Ottawa, Ottawa, Ontario, Canada
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
*Correspondence to:
Department of Biology
30 Marie Curie
Ottawa, ON
K1N 6N5
Canada
Phone 613-562-5800 x6004
Fax 613-562-5486
e-mail [email protected]
Copyright © 2017 by the American Physiological Society.
2
28
The field of transport physiology was fundamentally changed by the discovery of transport
29
proteins that are specific for small neutral molecules such as urea, water, ammonia and CO2.
30
The identification of first urea transporters (12), then aquaporins or water channels (1), and most
31
recently ammonia transporters (8)(that may also facilitate the movement of CO2), challenged the
32
long-held view that membranes were relatively permeable to these small neutral molecules,
33
which were thought to traverse a lipid bilayer directly, driven by a diffusion gradient. By
34
contrast, the involvement of specific transport proteins suggests a far higher capacity for control
35
over the movement of these molecules, as is the case for ions that move across membranes via
36
protein transporters or channels. The ammonia-transporting Rhesus-associated (Rh)
37
glycoproteins are particularly interesting in this context because they function in the transport of
38
either or both ammonia gas NH3, a small neutral molecule, and the ammonium ion NH4+ (2), the
39
two forms of ammonia that are in equilibrium with each other (‘ammonia’ here is used to denote
40
total ammonia, i.e. NH3 + NH4+). The mammalian Rh glycoproteins belong to solute transporter
41
family SLC42 and comprise the red blood cell-specific Rhag, as well as Rhbg and Rhcg, which
42
exhibit broader tissue distributions including prominent expression in the kidney (9). In the
43
kidney, Rhbg and Rhcg play a vital role in ammonia secretion and hence in the maintenance of
44
acid-base balance, which in mammals is largely dependent on renal bicarbonate ion reabsorption
45
and net acid excretion. Net acid excretion, in turn, reflects renal ammonia metabolism, the
46
balance between ammoniagenesis and ammonia excretion at the kidney (9).
47
The primary role of the kidney in maintaining acid-base balance in mammals is in fish
48
played by the gill (6). Moreover, the multifunctional gill also serves as a key site of ionic and
49
osmotic regulation and nitrogen waste excretion, with most fish (the sharks, skates and rays
50
being notable exceptions) excreting nitrogen waste predominantly as ammonia. With the
3
51
identification of Rh glycoproteins in fish (7), the scene was set for a paradigm shift in our
52
understanding of nitrogen excretion in fish and its integration with ion transport pathways and
53
acid-base balance (10), a shift in which epithelial transport of ammonia is viewed as a regulated
54
process rather being dependent on passive diffusion. In the ensuing years, increasing research
55
effort has focused on the branchial expression and regulation of Rh glycoproteins in a range of
56
fish species and in response to changes in environmental conditions. But what happens to
57
ammonia excretion when gill function is compromised, for example in species that emerge from
58
water onto land with a resultant collapse of gill surface area? Study of such species has revealed
59
interesting and often unexpected solutions to the problem of ammonia excretion in terrestrial
60
conditions, including induction of urea production and excretion of ammonia across the skin,
61
with localization of Rh glycoproteins to the skin (11). Hagfish present another situation in which
62
ammonia excretion at the gill may be compromised. These jawless fish are perhaps best known
63
for their ability to produce copious quantities of slime as an anti-predator strategy, but they are
64
also well known as scavengers that burrow into carrion on the ocean floor. Clifford et al. (4)
65
recognized the difficulties of this lifestyle with respect to branchial ammonia excretion –
66
inserting the head and branchial region into decomposing tissue high in ammonia is likely to
67
result in ammonia accumulation via gill Rh glycoproteins. They hypothesized that hagfish could
68
compensate for such ammonia accumulation by favouring cutaneous ammonia excretion across
69
the posterior region of the animal, which often remains exposed to seawater while the animal
70
feeds.
71
In support of this hypothesis, Clifford and colleagues (4) reported greater abundance of
72
Rhcg in skin from the middle and posterior regions of Pacific hagfish (Eptatretus stoutii) than
73
from the anterior region. Accompanying these regional differences in expression of ammonia
4
74
transport proteins were differences in the ammonia flux measured across excised pieces of skin –
75
ammonia excretion measured in vitro increased linearly for skin sections sampled from the
76
anterior to the posterior region of the animal. These data indicate that the capacity for cutaneous
77
ammonia excretion increases along the length of the animal, from snout to tail. In addition,
78
ammonia excretion across excised skin was increased by prior exposure of the animal to high
79
ambient ammonia levels, suggesting regulation of the cutaneous ammonia excretion pathway in
80
response to conditions where it might be favoured. Unfortunately, the authors were unable to
81
collect data on Rhcg protein expression in skin samples from animals exposed to high ammonia.
82
At the whole-animal level, a divided chamber approach was used to collect independent
83
measurements of ammonia flux for the anterior skin and gills versus the posterior skin. These
84
measurements revealed that while overall ammonia excretion was dominated by excretion via the
85
gills (and anterior skin), the contribution of posterior skin to overall ammonia excretion was
86
disproportionately increased in animals that had been exposed to high ambient ammonia levels.
87
Collectively, these data suggest a scenario in which loss of ammonia gained across the anterior
88
end of the animal when it burrows into decaying carrion is favoured by posterior excretion of
89
ammonia via the skin, with the additional possibility that cutaneous ammonia transport capacity
90
is enhanced by exposure to high ammonia conditions.
91
Solidifying this hypothesis will require additional study, in particular to quantify branchial
92
versus cutaneous surface area - a limitation of the study by Clifford and colleagues (4) was the
93
inability to partition ammonia flux into branchial and cutaneous components in a quantitative
94
fashion. Assessment of cutaneous and branchial Rh glycoprotein expression in response to
95
feeding and ambient ammonia exposure both individually and in combination also should be
96
explored. Enhanced cutaneous ammonia transport capacity in response to high ambient
5
97
ammonia levels could be a disadvantage if the entire surface of the hagfish (rather than just the
98
anterior end) is surrounded by carrion, a situation that requires consideration. Hagfish would
99
presumably benefit from mechanisms that limit ammonia gain when the anterior end of the
100
animal is embedded in decaying carrion, and some indications support this possibility (3, 4). For
101
example, plasma ammonia concentrations stabilize over time in hagfish exposed to high ambient
102
ammonia levels (3) and exposure of the anterior end of the animal to high ammonia levels has
103
little impact on plasma ammonia relative to exposing the posterior end of the animal (4). These
104
observations, coupled with reports of ammonia excretion occurring against an inwardly directed
105
ammonia gradient (3), suggest the existence of an active ammonia excretion mechanism that has
106
yet to be identified. Finally, unanswered questions remain with respect to the transport capacity
107
and/or permeability of hagfish skin for gas transfer. The data of Clifford and colleagues (4)
108
suggest that a small but physiologically relevant component of ammonia excretion occurs across
109
the skin of Pacific hagfish. At the same time, however, the skin in this species does not appear to
110
play a significant role in oxygen uptake, likely owing to its thickness (70-100 µm) and the
111
potential for the formation of boundary layers depleted in oxygen (5). Localization of Rhcg to
112
the basal portion of the epidermis (4) may facilitate ammonia movement out of the blood into the
113
epidermis, but does not address its movement from there to the surrounding water.
114
Measurements of cutaneous CO2 excretion, which would also provide insight into the practicality
115
of the skin as a gas exchange site, appear to be lacking for hagfish. In short, the observations of
116
Clifford and colleagues (4) that hagfish may gain ammonia across the gills when the head is
117
buried in carrion only to lose it across the skin at the tail end of the animal are intriguing not least
118
because of the many questions that remain to be addressed.
6
119
Acknowledgements
120
121
122
123
The author’s work is supported by Discovery and Research Tools & Instruments grants
from the Natural Sciences and Engineering Research Council of Canada.
7
124
References
125
126
1. Agre, P., Preston, G. M., Smith, B. L., Jung, J. S., Raina, S., Moon, C., Guggino, W. B.,
127
and Nielsen, S. (1993). Aquaporin CHIP: the archetypal molecular water channel. Am. J.
128
Physiol. 265, F463-F476.
129
2. Caner, T., Abdulnour-Nakhoul, S., Brown, K., Islam, M. T., Hamm, L. L., and Nakhoul,
130
N. L. (2015). Mechanisms of ammonia and ammonium transport by rhesus-associated
131
glycoproteins. Am. J. Physiol. 309, C747-C758.
132
3. Clifford, A. M., Goss, G. G., and Wilkie, M. P. (2015). Adaptations of a deep sea scavenger:
133
High ammonia tolerance and active NH4+ excretion by the Pacific hagfish (Eptatretus
134
stoutii). Comp. Biochem. Physiol. A 182, 64-74.
135
4. Clifford, A. M., Weinrauch, A. M., Edwards, S. L., Wilkie, M. P., and Goss, G. G. (2017).
136
Flexible ammonia handling strategies using both cutaneous and branchial epithelia in the
137
highly ammonia tolerant Pacific hagfish. Am. J. Physiol. In press.
138
5. Clifford, A. M., Zimmer, A. M., Wood, C. M., and Goss, G. G. (2016). It's all in the gills:
139
evaluation of O2 uptake in Pacific hagfish refutes a major respiratory role for the skin. J.
140
Exp. Biol. 219, 2814-2818.
141
6. Evans, D. H., Piermarini, P. M., and Choe, K. P. (2005). The multifunctional fish gill:
142
dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of
143
nitrogenous waste. Physiol. Rev. 85, 97-177.
144
145
7. Huang, C.-H. and Peng, J. (2005). Evolutionary conservation and diversification of Rh
family genes and proteins. Proc. Natl. Acad. Sci. USA 102, 15512-15517.
8
146
8. Marini, A.-M., Matassi, G., Raynal, V., André, B., Cartron, J.-P., and Chérif-Zahar, B.
147
(2000). The human Rhesus-associated RhAG protein and a kidney homologue promote
148
ammonium transport in yeast. Nature Genetics 26, 341-344.
149
150
151
152
153
154
155
9. Weiner, I. D. and Verlander, J. W. (2014). Ammonia transport in the kidney by Rhesus
glycoproteins. Am. J. Physiol. 306, F1107-F1120.
10. Wright, P. A. and Wood, C. M. (2009). A new paradigm for ammonia excretion in aquatic
animals: role of Rhesus (Rh) glycoproteins. J. Exp. Biol. 212, 2303-2312.
11. Wright, P. A. and Wood, C. M. (2012). Seven things fish know about ammonia and we
don't. Respir. Physiol. Neurobiol. 184, 231-240.
12. You, G., Smith, C. P., Kanai, Y., Lee, W.-S., Steizner, M., and Hediger, M. A. (1993).
156
Cloning and characterization of the vasopressin-regulated urea transporter. Nature 365,
157
844-847.
158