UREA RETENTION MECHANISMS IN THE BRANCHIAL EPITHELniTM OF A MARINE ELASMOBRANCH, THE S P D N DOGFISH (SQUAXUS ACANTHL4S) A Thesis Presented to The Facdty of Graduate Studies of The University of Guelph by GLENN ALEXANDER FINES In partial fiilfillment of requirernents for the degree of Master of Science July, 2000 O Glenn A. Fines, 2000
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UREA RETENTION MECHANISMS IN THE BRANCHIAL EPITHELniTM OF A
MARINE ELASMOBRANCH, THE S P D N DOGFISH (SQUAXUS ACANTHL4S)
A Thesis
Presented to
The Facdty of Graduate Studies
of
The University of Guelph
by
GLENN ALEXANDER FINES
In partial fiilfillment of requirernents
for the degree of
Master of Science
July, 2000
O Glenn A. Fines, 2000
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ABSTRACT
T m MECECANISMS OF UREA RETENTION IN BRANCHIAL EPITHELIUM OF ELASMOBRANCHS
Glenn Alexander Fines University of Guelph, 2000
Advisors: Dr. J.S. Baliantyne and Dr. PA. Wright
The retention of hi& concentrations of urea in the tissues of marine
elasmobranchs is the key to their osmoregulatory strategy. The rnechanisms responsible
for the low urea permeability of the gill epithelium nom the sphy dogfïsh (Squalus
acanthias) were investigated using enriched basolateral membrane vesicles (BLMV).
Urea uptake &=IO IIM) was sodium dependent and inhibited by phloretin (Iso=0.08
mM) and urea analogs (e.g. thiourea and methylurea). Much of the impermeability of the
impermeability of the BLMV may be due to the very high cholesterol content apparent
fiom the high cholesterol to phospholipid ratio (3.68). The high phosphatidylcholine and
low polyunsaturated fatty acid Levels are also likely to confer increased order to the
bilayer membrane, making it less fluid and less permeable.
Taken together, these findings indicate that low urea permeability in the dogfish
gill is primarily due to an active urea transporter that returns urea to the blood against the
concentration gradient and a unique lipid composition that minimizes diffusion urea
across the basolateral membrane.
ACKNOWLEDGEMENTS
1 gratefidly achowledge the f3nancial support of the Natural Sciences and
Engineering Research Council and the Huntsman Marine Science Centre. I would like to
thank my advisors, Dr. Jim BalIantyne and Dr. Pat Wright for their support and gïving me
the chance to work on this great project I a h want to thank Dr. Glen Van der Kraak for
serving on my advisory cornmittee and all of his helpful comments on my thesis. 1 thank
dl of my labmates (Jason, Natasha, Andy, Marc, J o ~ , Andrea, and Dave) for listening to
my incessant chattering about urea transporters and throwing great parties. 1 wouid like
to thank my family for their support. Finally, 1 wodd Like to thank Jennifer for her
support and encouragement.
TABLE OF CONTENTS
........................................................................................................ Açknowledgements i
.. Table of Contents .......................................................................................................... 11 ... ............................................................................................................... List of Tables iir
.............................................................................................................. List of Figures iv
.............................................. General Introduction .. .................................................. 1
Chapter 1 Active urea transport and an unusual basolateral membrane composition in the gills of a marine elasmobranch .......................................................... 11
Chapter 2 Lipid Composition of the Basolateral Membrane in Gill Epithelium of the ..................................................................... Spiny Dogfish (Squalus acanthias) 56
................................................................................................................... Appendix I 95
.................................................................................................................. Appendiv II 96
LIST OF TABLES
Table 1.1. Marker enzyme specific activities and magnitude of purification of basolateral membrane ............................. ,.. ................................................. -24
Table 1.2. Total activity of marker enzymes, percent recovery, and percent contamination in the final basolateral membrane vesicle preparation ............. 25
Table 1.3. Percentage of the basolateral membrane fraction as resealed and the orientation of the vesicles ............................................................................... -26
Table 1.4. Percentage of phospholipid types and total phospholipid and cholesteroi in the basolateral membrane of gill epithelium fiom the spiny dogfish,
Table 1 S. Cho1esterol:phospholipid ratios of representative species fkom difTerent .............................................................................................................. phyla.. .43
Table 1.6 Cornparison of cholesterol to protein ratios in the basolateral membrane of the gill epithelium fiom the spiny dogfïsh and a marine and fieshwater
Table 2.1. Cumulative percentages of individuai fatty acids in gill basolaterd plasma membrane fiom Squalur acanthias ..................................................... 63
Table 2.2. Percentages of individual fatty acids in the major phospholipids fiom the ................................. gill basolateral plasma membrane of Squalus acanthias 64
iii
LIST OF FFGURES
Figure 1.1 Filter optirniration for autofluorescence and non-specinc binding of 14c-
urea for use in urea transport rapid filtration experiments. Blanks involve filtering the radioactive mixture only through the filter. Controls involve adding radioactive mixture to diluted vesicles and immediately filtering through the filter. (Mean t SE, n=2) ............................................................... 28
Figure 1.2 a. Rates of urea uptake at variable urea concentrations, by BLMV fiom the gill of the dogfkh, SquaZus acanthias. (Mean t SE, n=8). b. Expansion of the low end of the [urea] range fiom a. The regression is y = 0a07741n(x) + 0.01 l6 ,Z = 0.9064 (Mean & SE, n=7) c. Lineweaver-Burke transformation of the effect of variable [urea] on uptake rate by BLMV. The regression is y=2.96 12t29.8 lx, 2=0.9778 (Mean f SE, n=7) ................. 30
Figure 1.3 Dose dependent phloretin inhibition of urea uptake in BLMV fiom the gill of the dogfish, Squalus acanîhias (Ise = 0.08 mM) (Mean t SE, n=5) ...... 33
Figure 1.4 Inhibition of urea uptake in BLMV by the urea analogues, acetamide, thiourea, N-methylurea, and NPTU (3 70 mM). (Mean & S .E., n=3). ............ ..3 5
Figure 1.5 ATP (10 mM) stimulation of urea uptake and effects of ouabain (1 mM) and NEM (1 mM) in BLMV fiom the gill of the dogfkh, Squalur acanthias (Mean + SE, n=8). * significant merence fiom control (paired t-test,
Figure 1.6 Rate of urea uptake in BLMV from the gill of the dogfish, Squalur acanthias, in the presence of physiologically oriented sodium (225 mM outwardly directed) or potassium gradients (225 mM inwardly directed) (Mean f SE, n=6). * sipifkant dserence fiom control (paired t-test, pcO.005) ** significant difference between sodium and potassium (paired t-
Fig. 1.8 A mode1 for the mechanism of reduced urea permeability of the dogfish gill basolateral membrane due to cholesterol. The cholesterol (red) contributes to a more tightly packed phospholipid bilayer membrane (yellow) thereby
... physically reducing the permeability of the basolateral membrane to urea.. 54
GENERAL INTRODUCTION
The vertebrate class Chondrichthyes (the cartilaginous fishes) is an ancient
lineage that contains two extant subclasses, the Elasmobranchii (sharks, skates, and rays)
and the Holocephali (ratfïsh or chimaeras) (Pough et al. 1996). The Elasmobranchii
evolved fiom the early chondrichthyans, which first appeared during the early Devonian,
approximately 400 million years ago (mya). The most recent radiation of elasmobranchs
appeared by the early Triassic (245 mya), with living families having evolved by the
Jurassic (208 mya) and extant genera appearing in the Cretaceous (144 mya) (Pough et al.
1996). Modem elasmobranchs typically occur in marine environments, dthough some
ascend rivers beyond tidal influence and some are permanent inhabitants of fieshwater
(i.e. Potomoîrygon spp.). The most fascinating characteristic of the elasmobranchs,
however, is the presence of elevated bIood levels of urea (350 to 600mM) (Robertson
1989). In most vertebrates urea is a waste product of amino acid catabolism and is
excreted fiom the body as rapidly as it is produced. Retention of such levels of urea in
elasmobranchs is therefore of considerable interest and has been under investigation for
over a century.
Discoverv of urea in elasmobranchs
Stadeler and Frérichs (1858) were the fist to discover the presence of "colossal
quantities" of urea in extracts fiom the muscle of Raja batis, R. clmata, and the dogfïsh
Scyllurn canicula (=Scylorhinus canicula). Their examination of other animals,
including teleosts and lamprey (Peh.omyzon sp.) revealed only traces of urea. Stadeler
(1 859) extended this observation to the spiny dogfish Spinm acanthias (=Squalus
acanthias) and the torpedos Torpedo marmorata and T. ocellata, and this was later
codbmed by Schultze (1 861) and Rabuteau and Papillon (1873). Knikenberg (1 88 1,
1 886, 1887, 1888) was the fïrst to undertake a systematic examination of the distribution
of urea in vertebrates and demonstrated that large amounts of urea were present not only
in the Selachii and Batoidei (=ElasmobranchÜ), but in the Chimaeroidei (=Holocephali)
as weU, although not in the lungfish (Neoceradohcs). This was an important observation
because of the close relation between the Elasmobranchii and Holocephali. Although the
lungfkh was thought to be extremely primitive, it was more closely affiliated with the
higher bony fishes. This suggested that the presence of elevated blood urea levels was an
evolutionary adaptation shared by ail Chondrichthyans. However, the function of urea in
these organisms and the physiological mechanisms by which the observed uraemic state
(pathological elevation of blood urea in mammals) is brought about, remained to be
described.
Earlv investigations of the function of urea in elasrnobranchs
The involvement of urea in the production of the electric discharge of the electric
organ of Torpedo ocellata was the first proposed function for urea in an elasrnobranch
(Gréhant and Jolyet 1891). They had observed that an increase in the urea content of the
electric organ tissue accompanied the electric discharge. Two years later Rohmann
(1893) reexamined the Gndings of Gréhant and Jolyet (189 1) and concluded that neither
urea nor any other nitrogenous compounds were involved in the production of electricity.
Baglioni (1906% 19 l7b) later confïrmed this by analysing various compounds in the
muscle, electric organ and senun of torpedo. His analyses revealed great difiibility of
urea and its nearly uniform distribution throughout the body of T. ocellata, including the
electric organ.
Another theory for the role of urea in elasmobranchs that was popdar for over 20
years at the tum of the century was that urea was essential for cardiac hct ion. Straub
(1901) found that 3.4% NaCl (the same osmotic pressure as elasmobranch blood) could
not keep the heart beating in good condition. Baglioni (1905) confinned Straub's
observation, having examined the effects of various NaCl solutions upon the heart and
concluded that urea was absolutely essential for cardiac activity. Baglioni (1905)
inferred that all elasmobranch tissues required urea for proper h c t i o n with the ideal
mixture being 2% NaCl and 2.2% urea. Further observations by Baglioni (1 9O6b)
demonstrated that the urine of elasmobranchs contained only low levels of urea, which he
believed to confirm his fïnding that urea was essential to life, by neutralizing the harmfùi
effects of high NaCl levels in the blood. Botazzi (1906), however, cnticized Baglioni's
experiments based on the fact that the red blood cells of elasmobranchs haemolysed in a
urea solution isotonic with 1.3-1.6% NaCl. He argued that a 3.4% NaCl solution was
hypertonie for the elasmobranch heart if the heart was equally permeable to urea because
the total osmolarity of the heart would be made up of both ionic and urea components,
not just ionic components. The key to a urea fiee perfusion medium eluded discovery
(Fühner 1908, De Meyer 19 10, Bornpiani 19 l3), and Baglioni (1 9 Ua) reasserted his
original view that urea was necessary for the maintenance of physiological activity in the
heart and other tissues. He even went so far as to classi@ urea as a hormone, according
to the definition of Bayliss and Starling (1902).
Frédéricq (1922) was the fist to attempt to disprove Baglioni's theory. He began
by confïrmïng that 3.5% NaCl was incapable of maintaining contractility in the ScyZZium
heart, whereas 2% NaCl and 2% urea was. However, he ccntinued by experimenting
with weaker salt solutions and found a urea-fkee solution would maintain a heart beat for
a long t h e when potassium and calcium was present in definite proportions. Frédéricq
concluded that urea plays an important role with regard to the osmotic pressure of the
blood, but is not important osmotically or chernically with regard to the tissues, which are
fkeely permeated by i t Simply stated the current thinking of the time held that urea
fünctioned in an osmotic role across the branchial lamellae, which are Unpermeable to it,
while within the organism it is essentially inert (Smith 1936).
Osmotic role of urea in elasmobranchs
The theory that urea was used for an osmotic hc t ion was hrst alluded to during
the early studies of elasmobranch physiology (Rodier 1 899, 1900, Frédéricq 19O4), but
was not M y embraced until much later (Frédéricq 1922, Duvall and Portier 1923, and
Smith 193 1). It was revealed that elasmobranch blood is normally hypertonie to seawater
@uvall and Portier 1923) and Duvall(1925) estimated that on average, urea is
responsible for 44% of the total osmotic pressure of the blood, and therefore contributes
significantly to the maintenance of the hypertonicity of the blood. This lead to the
conclusion that urea is osmotically important with regard to the extemal environment
(Duvall 1925). The analysis of the blood of fieshwater elasmobranchs revealed that the
chIoride content was 25% lower than in marine elasmobranchs (Thorson et al. 1 967).
Urea content is also rnuch lower, accounting for half of the 50% reduction in total
osmotic pressure of the blood. This discovery led to the conclusion that urea is
physiologically involved in osmotic adaptation, particularly to a marine habitat (Smith
193 1). This has been the accepted function for urea ever since and research has focused
on the mechanisms responsible for enabling the use of urea in this way.
Phvsiolo@cal mechanisms of urea retention
Znitially it was believed that the "rnechanism" responsible for the elevated blood
urea Zevels found in elasmobranchs was rend insufïiciency, or the inability of the kidney
to remove urea fiom the blood (von Schroeder 1890). This theory was short-lived,
however, and was discarded once it was realized that the elevated blood urea levels
played a physiological role (the exact role had yet to be elucidated). Thus began the
search for the mechanisms responsible for urea retention in the elasmobranchs.
It was quickiy determined that the elasmobranch kidney played an active role in
urea retention. Baglioni (1906~) reported that the urea content of elasmobranch urine
rarely rose above one third of that of blood. It was later calculated that approximately
90% of the urea filtered by the glomedus is reabsorbed by the kidney tubule (Clarke and
Smith 1932). The exact site within the kidney and the mechanism responsible for this
reabsorption are uncertain due to conflicting and incomplete evidence for active andor
passive urea reabsorption. Evidence for an active mode of urea reabsorption in the
elasmobranch kidney indudes: 1) the fractional excretion of urea is 0.5% under normal
conditions (similar to active glucose reabsorption) (Kempton 1953), 2) urea reabsorption
is iso-osmotic (Kempton 1953), 3) 95% of filtered urea is reabsorbed, whereas only 35%
of the urea analog thiourea is reabsorbed (Boylan 1967), 4) ~ a + and urea are reabsorbed
at a fked ratio of 1.6 moles of urea per mole of ~ a ' (Schmidt-Nielsen et al. 1972), and 5)
both phloretin and chromate (inhibitors of urea transport in the toad bladder) inhibit urea
reabsorption in the dogfish kidney (Hays et al. 1977). These finding have led to the
proposai of an active urea reabsorption mechanism (Smith 193 1, Kempton 1953,
Schmidt-Nielsen et al. 1972) with loop II of the elasmobranch nephron being implicated
as a possible site for active sodium-linked urea reabsorption (Stolte et al. 1977).
However, there is conflicting evidence which suggests the possibility for the
involvement of a passive urea transport mechanism in the reabsorption of urea in the
elasmobranch kidney, including: 1) failure to detect a transport maximum for urea despite
membrane population. This group of resealed vesicles is composed of 9.41% inside-out
and 25.59% right side-out orientation (Table 1.3). Freezing and thawing of the final
BLMV preparation resulted in a decrease in the percentage of resealed vesicles of both
orientations (Table 1.3), despite the passage of the membranes through a 23-gauge needle
&er thawing. The volume of the BLMV was relatively large, 8.60 f 1.78 p l h g protein,
providing additional confirmation of vesicle resealing.
Filter Control Test
None of the nIters tested exhibited any appreciable autofluorescence in the
scintillation cocktail used (Fig. 1.1 ; Blanks). The optimal filter type for use with I4c-urea
was found to be the polycarbonate Isopore filter from Mïllipore (Fig. 1.1). This filter
produced consistently low background counts due to non-specific binding of 14c-urea to
the filter. This accounted for approximately 10% of the total counts in the urea uptake
assays, similar to previous studies (Hopfer et al. 1973).
Concentration Dependence of Urea Uvtake bv BLMV
Urea uptake by BLMV was measured over a range of urea concentrations in the
incubation medium revealing two components of uptake (Fig. 1.2a). At hi&
concentrations (15 - 370 mM) urea uptake is linearily dependent upon the urea
concentration (Fig. 1.2a). However, at low urea concentrations (1 - 15 mM) urea uptake
exhibits saturation-like kinetics (Fig. 1.2b). When these data are transformed using a
Lineweaver-Burke plot, it is revealed that urea uptake at urea concentrations of 1 - 15
mM has a Km of 10.07 mM and V,, of 0.338 poVhr/mg protein (Fig. 1.2~).
Figure 1.1 Filter optimization for autofluorescence (blanks) and non-specific binding of
14c-urea (controls) for use in urea transport rapid filtration experiments. The blanks
represent fïiters only in scintillation cocktail. The controls involve adding radioactive
mixture to pre-diluted vesicles followed by immediate filtration through the filter. (Mean
k SE, n=2).
Figure 1.2 a. Rates of urea uptake at variable urea concentrations, by BLMV fiom the
giii o f the dogfïsh, Squaluî acanthias. (Mean k SE, ~ 8 ) . b. Expansion of the low end of
the [urea] range fiom A. The regression is y = 0.077Ln(x) + 0.0 12,8 = 0 .go6 (Mean + SE, n=7) c. Lineweaver-Burke transformation of the effect of variable [urea] on uptake
rate by B L W . The regression is y=2.96 + 29.8 lx, 1?=0.978 (Mean f SE, ~ 7 ) .
O 50 100 150 200 250 3 0 0 3 5 0 4 0 0
Urea concentration (rriM)
Inhibition of Urea Uptake by BLMV
Urea uptake by BLMV demonstrated sensitivity to the non-competitive inhibitor
phloretin (Fig. 1.3). Phloretin produced a dose-dependent inhibition of urea uptake by
BLMV, with 50 % inhibition occurring at a concentration of 0.08 mM. The use of urea
analogs demonstrated competitive inhibition of urea uptake by BLMV. N-methylurea
and NPTU significantly reduced the rate of urea uptake (pc0.05; Fig. 1.4). Thiourea and
acetamide also tended to reduce the rate of urea uptake although this decrease was not
significantly different fiom the control. The sulfhydryl reagents pCMBS, PMSF, and
NEM did not show any inhibition of urea uptake by BLMV (data not shown).
ATP Deuendence and Inhibition of Urea Uptake by BLMV
Urea uptake was signincantly stimulated by the addition of ATP to the incubation
medium @<0.05; Fig. 1 S). However, upon the addition of ouabain the rate of urea
uptake by BLMV decreased to control levels (Fig. 1.5). The addition of NEM had no
effect on the ATP stimulated urea uptake (Fig. 1.5).
Cation De~endence of Urea Uptake by BLMV
There was no significant difference between urea uptake in medium containing
ody sodium or only potassium ions with no concentration gradient present (Fig. 1.6).
When urea uptake was measured in the presence of a potassium concentration gradient
there was also no significant change in the rate of urea uptake by BLMV. However,
when a sodium concentration gradient was present during urea uptake measurernents, the
'Signincantly difEerent fkom Fresh Water Arctic Char @<0.005) b~ignificantly different fiom Salt Water Arctic Char @<O.OS) CSignincantly dBerent fiom Salt Water Arctic Char @<0.000005) * ~ i ~ n i n c a n t l ~ different nom Winter Flounder (pc0.0005)
DISCUSSION
Methodolow
The method (Perry and Flik 1988) used to prepare basolateral plasma membranes
Çom the gill epithelium of the spiny dogfish (Squulus acanthias) yielded a specific
enrichment of ~ a f , K'-ATP~s~ indicating selective isolation of basolateral plasma
membranes. Although there was only minor contamination (6%) of membranes fiom
the endoplasrnic reticulum, mitochondria and nucleus, this may have led to a slight
underestimation of urea uptake by the gill BLMV. The final recovery of ~ a * , lC'-~TPase
activity (6.2%) is consistent with previous studies (Perry and Flik 1988), while the
vesicle orientation (IO=9.4%; RO=25.6%) and resealing efficiency (35%) were
somewhat lower than reported values for eel (IO=33%; RO=23%; resealing efficiency
56%) (Flik et al. 1985). The volume measurements c o b e d that the vesicles
successfblly resealed. The decrease in resealing efficiency afier fieezing and thawing of
the final BLMV preparation suggested decreased integrity of the membranes and
therefore onl y fieshly prepared BLMV were used for transport experirnents. Significant
t h e was saved by omitting the g d perfüsion step, used to clear the gills of red blood
cells (Perry and Flik 1988), as there are no urea transporters present in elasmobranch
erythrocyte plasma membranes (Carlson and Goldstein 1997).
Urea Transport
The measurement of urea uptake by enriched BLMV revealed saturation kinetics at
low urea concentrations (KmlO.1 mM, Vm,=0.34 pmoVh/mg protein), suggesting the
presence of carrier-mediated urea transport. The low Km, relative to the urea
concentration in the blood, indicates that the transporter has a relatively high affinity for
urea. This implies that the putative urea transporter acts to "scavenge" intracelldar urea,
actively returning it to the blood and thereby maintaining a low urea concentration within
the gill epithelial cells. The effects of several known inhibitors of urea transport were
examined to M e r characterize the saturable component of urea uptake by the BLMV.
Inhibition by the non-competitive inhibitor phloretin is diagnostic of both facilitated and
secondary active, urea transport systems Wato and Sands 1998, Levine et al. 1973, Smith
and Wright 1999, Walsh et al. 1994). The dose-dependent inhibition of urea uptake in
shark gill BLMV by phloretin in this study is consistent with a previous study on the
isolated perfused dogfish head preparation (Part et al. 1998), which demonstrated that
phloretin &ion signifïcantly increased urea efflux across the gill. The urea analogues
N-methylurea and NPTU also signincantly inhibited urea uptake in shark gill BLMV.
Acetamide and thiourea had a slight, but non-signincant effect on urea uptake rates.
However, these results are inconclusive u t i l the analogue concentrations are optimized,
since studies have shown that under dif5erent conditions urea analogues may or may not
have statistically signincant effects on the branchial urea efflwc in the spiny dogfkh (I?W
et al. 1998, Wood et al. 1995). In mammalian inner medullary collecting duct,
methylurea and thiourea signi6cantly reduced urea permeability, while acetamide did not
(Chou and Knepper 1989). These results demonstrate that urea transporters in difZerent
tissues and animals exhibit dif3erent sensitivities to urea analogues. The inhibition of
urea uptake in shark gill BLMV by phloretin and urea analogues supports previous
hypotheses (Smith and Wright 1999, Wood et al. 1995) for a carrier-mediated urea
transport system in the dogfish shark gill.
Urea uptake is energy dependent in shark gill BLMV. The addition of adenosine
triphosphate (ATP) to the incubation medium sienif?cantly increased the rate of urea
uptake, whereas ouabain, a specific inhibitor of the enzyme N~~,K+-ATP~s~, returned the
rate of ATP-dependent urea uptake to control levels. In the presence of NEM, an
alkylating agent that binds selectively to sulfhydryl groups blocking V-type and P-type
ATPases (e-g. proton pumps) (Ehrenfeld 1998), ATP-dependent urea uptake was
unchanged. This ouabain sensitivity, but NEM insensitivity, of ATP-dependent urea
uptake in shark gill BLMV signifies that urea uptake was indirectly ATP dependent,
coupled to either the sodium or potassium gradients created by N~+,K+-ATP~s~. In the
presence of an outwardly directed sodium gradient, urea uptake was signincantly higher
relative to both the control and potassium gradient experiment. Taken together, these
data confirm that the saturable component of urea uptake in the shark gdi BLMV is due
to a ~ a + - c o u ~ l e d secondary active urea transporter, similar to the type found in the
mammalian kidney (Kato and Sands 1 998).
The dog£ïsh gill urea transporter functions in an antiport fashion (Fig. 1.7), similar
to the transporter described in the rat inner medullary collecting duct, but differs from the
putative sodium-linked urea transporter described fiom the dogfkh kidney (Schmidt-
Nielsen et al. 1972), which is thought to k c t i o n in a symport fashion. By using the
inwardly directed concentration gradient of ~ a + , the dogfkh gill urea transporter actively
pumps urea back into the blood fiom within the cell. This would decrease the
intracellula. urea concentration of the gill epithelial cells, reducing the concentration
gradient for urea across the apical surface of the cells and thus the rate of urea diffusion
or loss across the gill. Depending on the stoichiometry of the exchange, the shark can
Fig. 1.7 Schematic representation of the proposed Na+-coupled, active urea transporter
present in the basolateral membrane of the dogEish gill epithelium. 1 propose that the
active retum of urea to the blood reduces the intracellular urea concentration thereby
reducing the rate of urea diffusion across the apical membrane to the seawater.
(Thickness of the dashed arrows indicate the relative rate of urea diffusion.)
Seawater O mM Urea
Blood 370 mM Urea
Urea
1 I I I
Urea
7
Apical
Basolateral
Urea ~ a + K'
Save up to 5 ATP equivdents (the metabolic cost of synthesizing one urea molecule via
the ornithine-urea cycle including glutamine synthetase) for each irrea molecule returned
to the blood. In the dogfrsh kidney, sodium and urea are reabsorbed at a Itixed ratio of 1.6
moles of urea per mole of ~ a + (Schmidt-Nielsen et ai. 1972). The energetic cost of
transporthg ~ a + via ~a+,lK+-~TPase is 1 ATP for every 3 moles- If a similar ratio of
exchange for sodium and urea were involved in the gill as in the kidney, savings of 4.8
ATP equivdents per molecule of urea retumed to the blood would be achieved. This
means that the metabolic cost associated with urea transport in h e gill is low, but the
metabolic savings are significant.
Gill basolateral membrane composition
In the basolateral membrane of the gill f?om the spiny dogfîsh, SquaZus acanthias,
PC and PE were the main phospholipids, typical of most eukaryotic membranes. The
resulting PC/PE ratio was 1.72. PC stabilizes the membrane as it favors the formation of
a laminar bilayer, while PE destabilizes the membrane by keeping it close to the phase
transition between laminar and hexagonal (HII) phase conformaQions (Thurmond and
Luidblom 1997). In most temperate species the ratio of PC to PE is approxirnately 1.0.
This ratio can be altered according to the enivronmental and physiologica1 conditions
encountered by the organism. For example, cold-acclimated organisms (Arctic char,
molluscs) increase the proportion of PE in their membranes iin order to maintain
membrane fluidity at low temperature (Hazel 1995). This results im a PCPE ratio of less
than one (0.3-0.5). However, in elasmobranchs the cellular membranes face the opposite
problern, increased fluidity due to urea (Barton et al. 1999). Orne adaptation that has
evolved in elasmobranchs to deal with this effect of urea is the presence of
trimethylamine oxide (TMAO). TMAO counteracts the negative effects of urea on both
proteins (Yancey and Somero 1980) and phospholipid membranes (Barton et al. 1999).
Another strategy, which may work in conjunction with TMAO, is increasing the ratio of
PC to PE, which would provide additional stability to the membrane.
The very high cholesterol to phospholipid molar ratio (3.68) reported here is the
highest for native membranes (Table 1.5). Cholesterol decreases the permeability of
biological membranes to urea (Pu& et al. 1989) by inducing an increased order of the
phospholipid molecules that compose the bilayer membrane, allowing them to pack
closer together forming a tighter barrier (Mouritsen and Jmgensen 1994). Cholesterol,
when inserted in the appropriate place in the membrane, also increases membrane
permeability to oxygen (Dumas et al. 1997), therefore the elevated cholesterol levels in
shark gill basolateral membranes would not impair gas exchange. We propose that the
high cholesterol content of shark gill basolateral membrane provide a physical barrier that
retards passive loss of urea at the gill without affecting oxygen permeability (Fig. 1.8).
This hi& C:P molar ratio may also explain the low permeability of the shark gill to water
(Boylan 1967, Part et al. 1998) and sodium (Boylan 1967) relative to teleost fishes. Our
hdings also point to the possibility of similar structural modifications occurring in the
mammalian kidney tubule where urea permeability varies dong the nephron (Sands et al.
1997).
In Perspective
Based on the results of this study we propose that a unique combination of
physiological and structural mechanisms is at least in part responsible for the low urea
permeabiLity of the dofish shark gill. The marine elasmobranch gill is approximately 80
h e s less permeable to urea than the teleost gill resulting in a urea efflux of 270
poVkg/hr (Part et al. 1998). If the elasmobranch gill were as permeable to urea as the
rainbow trout gill, the resulting urea efflux would be immense (10,000 pol/kg/hr) (Part
et al. 1998), because of the enormous blood-to-water gradient. Ushg data from the
present study, it is possible to calculate the relative contribution of active urea transport
to the ciifference between observed and predicted (based on rainbow trout gill urea
permeability) urea efflux rates. The maximal velocity of urea uptake (Vrn~0.34
jmol/h/mg protein) was corrected for vesicle resealing (35%), vesicle orientation (27%
of resealed vesicles), membrane recovery (6.15%), protein level (9 mg/animal), and
animal mass (1.3kg). This results in a total rate of active transport of urea, back into the
blood fiom within the gill epithelium, of 535 ~ o V k g / h . This value is approximately 6%
of the difference in urea permeability between the rainbow bout and elasmobranch gill
(i.e. 10 000 - 270 p o v k g h ) . The remaining 94% may be in part, or in whole, due to
the elevated C:P molar ratio in the basolateral membrane. Thus we envision that the
primary role of the basolateral membrane is to substantially reduce the influx of urea into
the gill epithelial cells, thereby maintaining low intracellular urea concentrations at which
the urea transport system hct ions . The urea transport system actively transports urea
out of the epitheiial cells back into the blood maintaining low intracellular urea
concentrations. The low intracellular urea concentrations achieved by these
conplimentary mechanisms, leads to a reduced diffhion gradient for urea across the
apical membrane and thus a lower effective permeability of the gill to urea. One could
argue, therefore, that high cholesterol levels and active urea transport in the gill
basolateral membranes probably CO-evolved in elasmobranchs enabling the retention of
m a and its use as a key component of their osmoregulatory strategy, while minimising
the energetic cost. However, the hi& non-specific urea uptake by the BLMV suggests
that there are other mechanisms andor structures, particularly the composition of the
apical membrane, which may contribute signifïcantly to the overall low urea permeability
of the elasmobranch gill. Further studies of the elasmobranch gill are thus required to
completely resolve this issue.
Fig. 1.8 A mode1 for the mechanism of reduced urea permeabiiity of the dogfish gill
basolateral membrane due to cholesterol. The cholesterol (dark) contributes to a more
tightly packed phospholipid bilayer membrane (light) thereby physically reducing the
pemeability of the basolateral membrane to urea.
UREA
UREA
CHAPTERZ: A comparison of phospholipid and cholesterol composition of the
basolateral membranes from the gill epithelium of an elasmobranch, the dogfïsh
(SquaZus acan fhias)
INTRODUCTION
Elasmobranchs are unique among marine fish (except for the coelacanth) in that
they retain high leveb of urea (400-600 mM) io their tissues and body fluids for the
purpose of osmoregulation (Smith 193 6). Although urea retention provides an efficient
mechanism for osmutic balance, the chaotropic properties of urea pose another problem,
protein and phospholipid bilayer destabilization. The presence of trirnethylamine oxide
(TMAO) in the blood and tissues counteracts the destabilizing properties of urea on
protein (Yancey and Somero 1980) and membrane integrity (Barton et ai. 1999).
Modification of the phospholipid and fatty acid composition of cellular membranes is
also invoived in maintainhg membrane integrity of fishes under a variety of
environmental conditions (i.e. temperature and salinity) (Hazel and Williams 1990).
Phosphatidylcholine (PC) with a high content of saturated fatty acids (SFA) stabilizes
phospholipid bilayers, while phosphatidylethanolamine (PE) containing more unsaturated
fatty acids, destabilizes phospholipid bilayers in fish (Hazel and Landrey 1988a,b).
Elasmobranch mitochondrial membranes have signincantly higher levels of saturated
fatty acids and signincantly lower levels of polyunsahirated fatty acids (PUFA) relative
to other marine fishes such as the hagfïsh and winter flounder (Glemet and Ballantyne
1996). It has been suggested that these characteristics are adaptations to maintain
mitochondrial membrane i n t e e in the presence of hi& urea concentrations in the
tissues (Glemet and Ballantyne 1996), but other elasmobranch membranes have not been
examined.
The elasmobranch gill, like the gill of al1 fish, is involved in gas exchange,
ionoregulation, and osmoregulation. In addition to these vital fùnctions the elasmobranch
gill must also act as a barrier to urea loss. Compared to the teleost gill, the elasmobranch
gill is approximately 80 times less permeable to urea than the teleost gül (Part et al.
1998). The basolateral plasma membrane of the elasmobranch gli epithelium is in
contact with high levels of urea in blood on the extraceliular side and purportedly low
levels of urea on the intracellular side and may be the main permeabiiity barrier (Wood et
ai. 1995). In chapter one, we identified a ~a+-coupled active urea transport mechaaism
(K,=10 mM) in the basolateral membrane (BLM) of S. acanthiar gills that would act to
scavenge intracehlar urea and retum it to the blood, against the concentration gradient.
In addition, we reported that these same membranes had a cholesterol (C) to phospholipid
(P) molar ratio of 3.68, an extremely hi& value compared to other tissues and organisms
(Table 1.5). A high C:P molar ratio is characteristic of membranes with reduced
permeability to low molecular weight solutes, such as urea (Lande et al. 1995). Taken
together, the urea transport system and hi& C:P molar ratio would facilitate urea
retention in dogfïsh gills. It is also possible that the phospholipid and fatty acid
composition may contribute to the low permeability of the dogfkh gill to urea. The lipid
composition of various epitheiial tissues (Le. mammalian epidermis and trout intestine,
skin, and opercular membrane) has been demonstrated to innuence ~ a + and water
permeability @i Costanzo et al. 1983, Ziboh and Miller 1990, and Ghioni et al. 1997).
The purpose of this study was to characterize the phospholipid and fatty acid
composition of the basolateral plasma membrane fiom the dogfïsh gill epithelium. We
hypothesize that the phospholipid and fatty acid composition of this membrane, coupled
with the high C:P molar ratio, is in part responsible for maintaining membrane integrity
and contributhg to the extremely low permeability of the elasmobranch gill to urea.
METHODS AND MATERIALS
Ex~erimental animals
Spiny dogfish (Squalur acanthiar) were coliected by Otter Trawl in
Passamaquody Bay, New Brunswick fiom mid-July to the end of August 1999 and
maintained at the Huntsman Marine Science Centre in outdoor tanks under natural
photoperiod, and supplied with filtered seawater (lO°C). The dogfish did not feed in
captivity and were heId for a maximum of 10 days prior to experimentation.
Basolateral membrane @LM) preparation
BLM was prepared £kom epithelial tissue scraped fiom the gill arches. Tissue was
homogenized using a Dounce homogenizer in a hypotonie homogenization b a e r
containing (in mM): 25 NaCl, 1 dithiothreitol, 0.5 disodium ethylenedinitrilotetra-acetic
which accounts for 82% of the cumulative fatty acid composition. Saturated fatty acids
were the prevalent class of fatty acids (38 %) (Table 2.1).
In the BLM, PUFA was the largest class of fatty acids in the phospholipid PE
(Table 2.2). The main individual fatty acids were 20:4n-6, 20511-3, and 22:6n-3 totaling
39% (Table 2.2). PE had the highest levels of an unknown fatty acid (Table 2.2). This
unknown fatty acid was tentatively identïfïed as 16:3 or 16:4 based upon the retention
time relative to stearic acid (Ackman 1962). In PI, SFA and PUFA were the prevalent
classes of fatty acids (Table 2.2). This was due to the large proportions of 18:O and
20:4n-6 (Table 2.2). In PS, SFA and PUFA were the most abundant classes of fatty acids
(Table 2.2) due primarily to 18:0, 20:4n-6 and 22:6n-3 although the monoene 18: 1 was
also prevalent (Table 2.2). In PC, SFA was the dominant fatty acid class (Table 2.2) as a
result of the levels of 16:O (Table 2.2). The monoene 18: 1 was also prevalent in PC
equalling (Table 2.2). Monoenes comprïsed the largest class of fatty acids in SM (Table
2.2). This was due to the levels of 14:l and 24:l (Table 2.2). The saturated fatty acids
14:O and 16:O were also prominent in SM (Table 2.2).
Table 2.1 Cumulative percentages of individual fatty acids in gin basolateral plasma
membrane fiom Squalus acanthiasa
Fatty acid (mol %)
24: 1 1.59 10.19 Total SFA 38.11 I 1.70 Total Monoene 28.02 10.72 Total PUFA 34.88 I 1.61 Total n-3 PUFA 17.86 10.97 Total n-6 PUFA 17.02 k0.80 n-3/n-6 PUFA 1 .O5 I 0.05 Unsaturation indexC 186.48 I 56.58 Mean Chain lengt.hd 18.66 & 0.40
Values presented as mean f S.E.M. (n=8). bvnknown A (9.08 min) eluted between 17: 1 (8.76 min) and l8:O (9.32 min). 'Unsaturation index = Çrnim; where mi is the mole percentage and ni is the number of C-C double bonds in fatty acidi. %lean chain length = Xfci; where 6 is the mole hction and Ci is the number of carbon atoms in fatty acidi.
Table 2.2 Percentages of individual fatty acids in the major phospholipids fiom the gill
1.81 f 0.13 0.40 f 0.11 0.08 I 0.01 0.05 I 0.01 10.68 & 1.63
0.45 I 0-06 0.05 i 0.04 ND 0.50 I 0.25 28.99 rt 2.13 Total SFA 45.91 f 1.20 20.54 I 2.02 45.42 i: 4.87 45.06 I 6.62 38.39 2.34 Total monoenes 29.50 f 0.53 33.28 + 1.41 4.79 I 0.61 18.72 f 2.97 53.78 i: 1.91 Total PUFA 25.16 i: 1-57 47.90 I 2.70 51.15 I 4.75 37.64 k 4.27 8.78 I 1.81 Totaln-3 PUFA 14.48 I 0.87 23.78 I 1.55 14.37 i 1.55 21.27 f 2.65 4.97 i 0.92 Total n-6 PUFA 10.68 f 0.81 24.11 f 1.41 36.78 f 3.98 16.37 k 1.79 3.81 I 1.05 n-3/nd PUFA 1.38 & 0.07 0.99 i- 0.05 0.42 i: 0.05 1.30 1: 0.09 1.56 * 0.27 Unsaturation 144.18*6.38 253.67k11.84 219.31 118.23 193.43f23.09 91.29i8.01 index Mean Chain 17.91 i 0.05 19.33 i 0.11 19.34 I 0.14 19.24 I 0.16 19.27 1: 0.34
values are presented as means f S.E.M. (n=8). ND not detectable. bvnknown A (9.08 min) eluted between 17: 1 (8.76 min) and l8:O (9.32 min).
DISCUSSION
Phospholipid Composition
Zn the basolateral membrane of the dogfkh gili epithelium, PC and PE were the
m a i . phospholipids, typical of vertebrate membranes (Hazel and Williams 1990). These
phospholipids play an important role in regulating the fluidity and permeability of
biological membranes (Hazel and Landrey 1988a). PC stabilizes the membrane as it
favors the formation of a laminar bilayer, while PE destabilizes the membrane by keeping
it close to the phase transition between larninar and hexagonal (HE) phase conformations
(Thurmond and Lindblom 1997). The ratio of PC to PE can be used as an indication of
membrane fluidity and rnay be altered according to the environmental and physiological
conditions encountered by an animd (Hazel and Williams 1990). In rainbow trout this is
clearly demonstrated when the PC/PE ratio dropped fiom 1.71 to 0.78 within 8 hours of
cold acclimation (5OC) and rose fkom 1.3 1 to 2.0 by the second day of warm acclimation
(20°C) (Hazel and Landrey 1988a). Despite inhabiting cold waters (5-10°C), the PC/PE
ratio in dogfish gill basolateral membrane (1.72) is quite high and similar to values seen
in warm acciimated rainbow trout (1.71 @ 20°C) (Hazel and Landrey 1988a) and
European yellow eels (Anguilla anguillu, 1.72 @ 15'C) (Aciemo et al. 1996). The high
PC/PE ratio in the dogfish gill BLM may be a physiological response to the increased
membrane fluidity caused by the hi& levels of urea found within the tissues and body
fluids.
It is also possible that the relatively high PCPE ratio in the BLM contributes to
the low urea pemeability observed in the dogfish gill (Part et al. 1998, Wood et al.
1995). Diet-induced increases of the PC/PE ratio in the bmsh border membranes fkom
rainbow trout can be correlated with decreased sodium permeability, demonstrating that
the capacity for PC molecules to pack closer together than PE molecules results in the
reduction of membrane permeabiiity (Di Costanzo et al. 1983). Active urea transport and
high cholesîerol levels have also been implicated as possible mechanisms involved in
reducing the permeability of the giu to urea (Fines et al. in press). The hi& cholesterol
levels in the BLM of the elasmobranch gill epithelium contributes to the highest reported
cholesterol:phospholipid molar ratio (Table 1.4). Cholesterol decreases the permeability
of biological membranes to urea by induchg increased order of the phospholipid
molecules that comprise the bilayer membrane (Mouritsen and Jmgensen 2994). We
proposed that the high cholesterol levels allow this membrane to firnction as a physical
barrier to the passive loss of urea, appearing as reduced urea permeability. Thus, a high
PC/PE ratio may be complimentary to the high cholesterol levels and also contributes to
the low urea permeability of the dogfish gill.
Fattv Acid Composition
Cumulatively, the most prominent fatty acid class Çom the phospholipids of the
dogfish gill BLM was the saturated fatty acids (38.11%, Table 2.1), similar to the
intestinal mucosa of the marine teleost Anguilla anguilla (39- 17%, Acierno et al. 1996).
Phospholipids containhg saturated fatty acids form tightly packed bilayers with relatively
low membrane fluidity and permeability, while phospholipids containing unsaturated
fatty acids pack less tightly resulting in more permeable membranes (Chen et al. 1971).
The chain length of fatty acids is also important in determining the fluidity and
permeability of a membrane. Longer chain fatty acids (18:O) increase the order of
phospholipid bilayers more than shorter chah fatty acids (1 6:O) thus contributhg more to
overall membrane stability (Chen et ai. 197 1, b e l and Landrey 1988b). Several studies
have demonstrated that fatty acid composition affects membrane permeability including
studies on ~ a + and glucose permeability in the b w h border membrane of trout @i
Costanzo et al. 1983) and channel catfïsh (Houpe et al. 1997), respectively. These studies
clearly demonstrated that modification of the levels of SFA in the phospholipids fiom
these membranes alters the permeability to these two solutes. The high levels of SFA,
both l6:O and 1 8 :O, in the dogfish gill BLM are likely involved in maintaining membrane
stability and the reduction of overall membrane permeability. This is supported by the
fact that phospholipids of mitochondrial membranes fiom fish that do not retain elevated
urea levels (hagfish and flounder) contain significantly higher levels of PUFA when
compared to those of the Little skate, an elasmobranch (Glemet and Ballantyne 1996).
This reflects the adaptation of these membranes to the different intemal solute
environments that occur in each of these fish. This difference between elasmobranchs
and other marine fishes is even more evident when one considers that hagfkh and
flounder occur at the same temperature and salinity as the little skate.
The monoene 18: 1 was cumulatively, the second most prominent fatty acid in the
phospholipids fiom the BLM of the dogfish gill epithelium. This also contributed to the
hi& level of monoenes in the dogfish basolateral membrane, relative to other fishes
(hagfïsh and flounder mitochondria, Glemet and Ballantyne 1996; European eel and sea
Van Praag, D., Farber, S.J., Minkin, E., and Primor, N. (1987) Production of eicosanoids
by the kdlifïsh g u s and opercular epithelia and their effect on active transport of ions,
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Von Schroeder, W. (1890) Über die harstonbildung der hainsche, Huppe-Seyl. S. 14,
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Walsh, P., Wood, C.M., Perry, S.F., and Thomas, S. (1 994) Urea transport by hepatocytes
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Walsh, P.J., Heitz, M., Campbell, CE., Cooper, G.J., Medina, M., Wang, Y.S., Goss,
G.G., Vincek, V., Wood, C.M., and Smith, C.P. Molecdar identification of a urea
transporter in gill of the ureotelic gulftoadfish (Opsanus beta), J. Exp. Biol. in press.
Wood, C.M., Part, P., and Wright, P.A. (1995) Ammonia and urea metabolism in relation
to gill b c t i o n and acid-base balance in a marine elasmobranch, the spiny dogfïsh
(Squalus ucanthias), J. Exp. Biol. 198, 1545- 1558.
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(1987) Characterization and comparison of lipids in different squid nervous tissues,
Biochirn. Biophys. Acta 922,78-84.
Yancey, P.H. and Somero, G.N. (1980) Methylamine osmoregulatory solutes of
elasmobranch fishes counteract urea inhibition of enzymes, J. Exp. 2001. 212, 205-
213.
Zabelinskii, S.A., Brovtsyna, N.B., Chebotareva, UA., Gorbunova, O.B., and
Krivchenko, A.I. (1995) Comparative investigation of lipid and fatty acid composition
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APPENDIX 1
Schematic representation of the assay of the percentage of inside-out-oriented vesicles. Assuming that resealed vesicles are impermeable to either ATP or ouabain, then N~+,K+- ATPase activity of untreated vesicles reflects the leaky vesicle fiaction. The detergent digitonin (0.04% w/v) stimulates N~+,K+-ATP~s~ activity by permeabilizing the membrane, providing ATP and ouabain access to the interior of the resealed vesicles. The percent ciifference in activity between untreated and digitonin trerited vesicles represents the percentage of resealed vesicles. The percentage of resealed vesicles with an inside-out orientation is obtained by measuring the stimulation of ATP hydrolysis induced by K+ ions of untreated and digitonin-treated vesicles in medium containhg ~ a + and ouabain. During short incubations, K' ions will equilibrate across the membrane quickly while ouabain does not. The N~+,K+-ATP~s~ protein of inside-out vesicles is inaccessible to ouabain and when digitonin is added the membrane barrier for ouabain is removed. This results in a decrease in N~+,K+-ATP~s~ activity which represents the percentage of inside-out vesicles. The difference between total resealed vesicles and inside-out vesicles equals the percentage of right-side out vesicles.
Right-side Out Ouabain
ATP
APPENDIX II
Schematic representation of the assay for measuring vesicle volume. Vesicles are incubated with a radiolabeled probe, such as 3 ~ f l , and then sedimented. The accessible volume of the pellet is calculated nom the specific activity of the probe in the supematant and the total radioactivity in the ellet. The difference in accessible volume (pellet P space) for penneant probes like H20 and probes like 1 4 ~ - ~ ~ ~ - 4 0 0 0 that do not cross the huer membrane equals the volume of the vesicle.
d.p.m. x supematant volume)/supernatant d.p.m. Vesicle Volume = space - "c-PEG-4000 space)/mgprotein added