|Cholesterol, Plasma Membranes and Temperature Acclimation
|The body temperature of poikilotherms, including most fish, varies
with environmental temperature. Homeoviscous theory holds that compensatory
restructuring of cellular membrane lipid composition is central to successful
temperature acclimation. In this way, vital membrane physical properties
('fluidity'), and associated membrane functions (e.g., acting as
diffusion barriers or matrices for protein activity) can be maintained
in a physiologically permissive range. To investigate the role cholesterol
plays in thermally-induced membrane restructuring, I isolated plasma membranes
from several trout tissues and quantified cholesterol content (1).
The elevated cholesterol content seen with acclimation to the warmer temperature
is consistent with biophysical
studies indicating that cholesterol orders most bilayer membranes; thus,
incorporation of stabilizing cholesterol could offset the molecular disorder
introduced into the membrane by higher temperature (2). One particularly
intriguing aspect of these data is the high cholesterol levels in gill
plasma membranes relative to other tissues. This result provoked pursuit
of my current primary research interest: does cell membrane lipid composition
influence the functional properties of fish gills?
|Fish Gill Barrier Membranes, Cholesterol and Water
|Given their range of homeostatic functions and interfacial
disposition with the ambient environment, gills are unique among physiologically
active epithelia. Morphologically, gills feature amplified exchange surface
areas and minimal blood-to-water diffusion distances; characteristics thought
functionally important for, inter alia, oxygen uptake. However,
in freshwater fish these structural attributes come at a potentially significant
cost: increased osmotic water uptake (a situation that has been termed
the 'osmorespiratory compromise'). Especially since the discovery of the
aquaporins (protein water channels), the concept that specific lipid composition
could limit the water permeability of biological membranes has gained increasing
attention. Cholesterol is well known to limit transmembrane permeabilities
in model systems; therefore, I am very interested in the relationship between
membrane lipid composition (particularly cholesterol) and water permeability
in the barrier membranes of gills of freshwater fish. I have used isolated
ligated gill arches from trout and warmwater tilapia to demonstrate that
osmotic water uptake is both temperature dependant and influenced by
acclimation status; properties consistent with a lipid bilayer water
permeation pathway (3). Moreover, using isolated arches, I have
gathered evidence for the importance cholesterol plays in restricting gill
water permeability by: a) using a cholesterol-complexing, pore-forming
antibiotic (nystatin) to increase osmotic water uptake, and: b) employing
cyclodextrin (CD) to manipulate the cholesterol level, and water uptake
rates, of gill barrier membranes. I have also been using another cholesterol-complexing
antibiotic, filipin, as a fluorescent cytochemical marker for cholesterol
in gill surface membranes (right).
These studies represent a foundation for pursuing
more direct, molecular-level investigations of membrane structure/function
relationships; e.g., isolating purified gill barrier membranes and
characterizing their lipid composition, membrane physical properties and
permeability characteristics. Ultimately, isolated gill barrier membranes
represent a highly relevant biological model for exploring a variety of
basic and applied physiological questions. Examples range from the effects
of temperature, salinity and xenobiotics on gill interfacial membrane composition
and functional properties to the issue of whether water and oxygen permeability
are independently influenced by membrane lipid composition. I am also interested
in whether certain anthropogenic (e.g., icthyotoxins containing
saponins) and natural (algal) toxins interact in a lipid composition dependent
manner with gill barrier membranes.
|Gill Pillar Cells, Ectoenzymes and Signal Transduction
|In characterizing trout plasma membranes, the activities of two plasma
membrane enzymes, 5'nucleotidase (5'NT) and alkaline phosphodiesterase
(APD), were found to be elevated in tissue homogenates and isolated plasma
membranes from gills relative to other tissues. These enzymes are widely
used as markers for the apical plasma membrane domain of vertebrate epithelial
cells. My interest in isolating gill epithelial cell apical plasma (barrier)
membranes lead me to further characterize these enzymes in the gill. I
used histochemistry to localize both 5'NT and APD activities exclusively
to the pillar cells which form the central microvascular
network in the gill epithelial outfoldings or lamellae (4). Significantly,
there is no indication of either enzyme activity associated with the surface
gill epithelial cells. These results will be quite valuable for demonstrating
effective separation of vascular and epithelial plasma membranes when isolating
gill barrier membranes. Histochemical staining patterns were consistent
with an apical plasma membrane localization for both activities in pillar
cells - i.e., the enzyme activities were found at the membranes forming
the actual microvascular lining. The functional significance of this localization
is intriguing. Both 5'NT and APD are involved in nucleotide metabolism
and have been suggested to act as modulators in purinergic signal transduction,
and pillar cells are unique microvascular cells with apparent endogenous
contractile capacity. Could these enzyme activities reflect a mechanism
for local regulation of hemodynamics in gill lamellae, perhaps in response
to hypoxia or other environmental conditions? The same pillar cell localization
for 5'NT and APD has been seen in all species of fish examined (freshwater
trout and tilapia as well as seawater flounder and eel), suggesting that
any associated physiological functions may be broadly conserved. I plan
to pursue functional questions and continue structural investigations of
gill pillar cells using immunolocalization and biochemical approaches.
|Developmental Biology, Sex Differentiation, Smoltification
|Interests in developmental biology and endocrine physiology derive
from work on the ontogeny of a sexual dimorphism in axon numbers in the
laryngeal neuromuscular system of Xenopus (5). One project
initiated in these studies examined primary (gonadal) versus secondary
(laryngeal masculinization) sexual development in tadpoles. By chemically
arresting metamorphosis, we demonstrated that while the gonads differentiate
independent of thyroid hormone (TH), the ability of the larynx to be masculinized
by androgens depends upon exposure to TH (6). I would like to return
to this system to look specifically at cellular and molecular hallmarks
of maturation in the developing gametes of metamorphically blocked tadpoles.
This work is provocative from an evolutionary perspective in that it may
illuminate a mechanistic basis for neoteny - a phenomenon in some amphibian
species whereby larval forms become reproductively mature.
I also have background and interests in more
applied aspects of fish biology stemming from my experiences with aquaculture
as an extension agent in the Peace Corps and aspects of my master's degree
work - involving, for example, environmental influences on smoltification
(fresh- to seawater transformation) in anadromous salmonids (7).
|(1) Robertson, J.C. and Hazel, J.R. 1995. Cholesterol content
of trout plasma membranes varies with acclimation temperature. American
Journal of Physiology, 269: R1113-R1119.
|(2) Robertson, J.C. and Hazel, J.R. 1997. Membrane constraints
to physiological function at different temperatures: Does cholesterol stabilize
membranes at elevated temperatures? In: Global Warming - Implications for
Freshwater and Marine Fish. Eds. C. Wood and G. McDonald. pp. 25-49. Society
for Experimental Biology Seminar Series.
|(3) Robertson, J.C. and Hazel, J.R. 1999. Influence of temperature
and membrane lipid composition on the osmotic water permeability of teleost
gills. Physiological and Biochemical Zoology. 72: 623-632.
|(4) Robertson, J.C. and Hazel, J.R. 1998. 5'Nucleotidase and
alkaline phosphodiesterase activities in trout gill localize to endothelial
(pillar) cells. Journal of Experimental Biology, 201: 2011-2019.
|(5) Robertson, J., Watson, J. and Kelley, D. 1994. Androgen
directs sexual differentiation of laryngeal innervation in developing Xenopus
laevis. Journal of Neurobiology, 25: 1625-1636.
|(6) Robertson, J.C. and Kelley, D.B.1996. Thyroid hormone controls
the onset of androgen sensitivity in the developing larynx of Xenopus laevis.
Developmental Biology, 176: 108-123.
|(7) Robertson, J.C. and Bradley, T.M. 1991. Hepatic ultrastructure
changes associated with the parr-smolt transformation of Atlantic salmon
(Salmo salar ). Journal of Experimental Zoology, 260: 135-148.