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JEZ 865
504
THEP.JOURNAL
SILVA ET AL.
OF EXPERIMENTAL ZOOLOGY 279:504–508 (1997)
Transport Mechanisms That Mediate the
Secretion of Chloride by the Rectal Gland of
Squalus acanthias
PATRICIO SILVA,* RICHARD J. SOLOMON, AND FRANKLIN H. EPSTEIN
Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672
Division of Nephrology, Beth Israel Deaconess Medical Center and Joslin
Diabetes Center, Boston, Massachusetts 02215
ABSTRACT
The rectal gland of Squalus acanthias secretes chloride by a mechanism that has
been termed “secondary active transport” because it depends on the activity of Na-K-ATPase. As
currently described, chloride enters the cell across the basolateral cell membrane via the 2 chloride: sodium: potassium cotransporter. The energy for this electroneutral uphill movement of chloride and potassium is provided by the gradient for sodium directed into the cell. Present in the
basolateral cell membrane is Na-K-ATPase that maintains the gradient for sodium. A potassium
conductance, present as well in the basolateral cell membrane, recirculates the potassium. Chloride exits the cell across the luminal membrane via CFTR, the chloride conductance. This mechanism is widely distributed throughout vertebrates. This report reviews the experimental
observations that led to the current definition of the mechanism of chloride transport in the rectal
gland. J. Exp. Zool. 279:504–508, 1997. © 1997 Wiley-Liss, Inc.
Since the identification by Burger of the rectal
gland as the organ responsible for the extrarenal
excretion of salt in Squalus acanthias (Burger and
Hess, ’59) its study has served to define the nature of chloride transport and of the transport proteins that mediate it. The finding by Stoff that
the secretion of chloride by the rectal gland can
be stimulated by cyclic AMP or theophylline allowed the study of the mechanism of chloride
transpost and its regulation (Stoff et al., ’77). The
basic mechanism for the secretion of chloride by
the rectal gland was initially identified in isolated
perfused rectal glands stimulated with cyclic AMP
or theophylline. The observations that the secretion of chloride by the gland is inhibited by ouabain and furosemide, and is dependent on the
sodium, chloride and potassium concentration in
the perfusate provided the necessary evidence for
the first description of the secretory mechanism
(Silva et al., ’77; Solomon et al., ’77, ’78). Based
on that evidence, a mechanism for the transport
of chloride was proposed in which the uptake of
chloride across the basolateral cell membrane is
mediated by a sodium-chloride cotransporter. The
energy for this uptake is provided by the sodium
gradient directed into the cell and maintained by
Na-K-ATPase (Silva et al., ’77). This initial model
for the secretion of chloride by the rectal gland
provided the first testable hypothesis for the
© 1997 WILEY-LISS, INC.
transport of chloride across epithelial tissues. In
this review we summarize the data that support
the role of each of the different transport proteins
that mediate the secretion of chloride by the rectal gland.
SODIUM, POTASSIUM ACTIVATED
ADENOSINE TRIPHOSPHATASE
(NA-K-ATPASE)
The first description of Na-K-ATPase in the rectal gland was that of Bonting (’66). Na-K-ATPase
of the rectal gland has been used by many investigators to examine the characteristics of the enzyme (Cornelius and Skou, ’84; Esmann et al., ’79;
Hokin et al., ’73; Skou and Esmann, ’79). Hokin
purified rectal gland Na-K-ATPase to 90–95% homogeneity, inserted it in phospholipid vesicles and
determined that it transported sodium and potassium in a 3:2 stoichiometry, establishing, for the
first time, a link between the biochemical activity of the enzyme and the transporting characteristics of the sodium pump (Hokin et al., ’73).
Na-K-ATPase is localized on the basolateral membrane of the rectal gland cell (Eveloff et al., ’79;
Goertmiller and Ellis, ’76). Stimulation of chlo-
*Correspondence to: Patricio Silva, M.D., Division of Nephrology,
Joslin Diabetes Center, Boston, MA 02215.
RECTAL GLAND TRANSPORT MECHANISMS
ride secretion by cAMP and theophylline in isolated perfused rectal glands results in a six-to
seven-fold rise in ouabain-inhibitable oxygen
consumption (Silva et al., ’79). Simultaneously, intracellular sodium concentration falls while intracellular potassium rises, suggesting activation of
Na-K-ATPase (Lear et al., ’92; Silva et al., ’79).
Stimulation with cyclic AMP and theophylline results in an increase in 86Rb uptake (Marver et al.,
’86). Further support for activation of Na-K-ATPase during stimulation of chloride secretion
comes from the observations that ouabain binding is increased in slices of rectal gland or isolated
rectal gland cells (Marver et al., ’86; Shuttleworth
and Thompson, ’80; Silva et al., ’83). Whether
there is direct activation of Na-K-ATPase during
stimulation or the effect is due to an initial rise
in intracellular concentration of sodium has not
been completely established. The increase in ouabain binding seen in response to stimulation is
independent of transport activity; i.e., it is seen
in the absence of sodium or in the presence of furosemide, suggesting that the enzyme is activated
directly (Silva et al., ’83). In recent studies
Cornelius et al. report the in vitro phosphorylation of reconstituted shark (no species identified)
Na-K-ATPase by protein kinase A is associated
with increased maximal activity of the enzyme in
response to both sodium or potassium (Cornelius
and Logvinenko, ’96). This finding suggests that
activation of chloride transport by peptides that
activate adenylate cyclase could directly activate
the enzyme. However, this observation is contrary
to the previous report that phosphorylation by protein kinase A inhibits the activity of the enzyme
(Chibalin et al., ’92).
SODIUM: POTASSIUM: 2 CHLORIDE
COTRANSPORTER
Studies in vesicles obtained from basolaterally
enriched plasma membranes prepared from rectal gland provided direct evidence for furosemidesensitive coupled sodium and chloride fluxes
across the plasma membrane of the rectal gland
(Eveloff et al., ’78). Potassium was found to be
needed for the uptake of sodium, and chloride for
that of rubidium, indicating that the cotransporter
for sodium and chloride required the presence of
potassium (Hannafin et al., ’83). Further studies
by these investigators demonstrated that the uptake of chloride into rectal gland plasma membrane vesicles required sodium and potassium and
was inhibited by bumetanide, supporting the hypothesis that the uptake mechanism is a sodium-
505
potassium-chloride cotransporter (Hannafin and
Kinne, ’85). Thus the model for the transport of
chloride by the rectal gland included a sodium:potassium:chloride cotransporter. This cotransporter has recently been cloned, first by Xu
et al. (’94) and then by Gamba et al. (’94). The
stoichiometry of the cotransporter was thought to
be 2 chlorides for every sodium because the gradient favoring the entry of sodium into the cell is
three times greater than that opposing the entry
of chloride (Silva et al., ’77), and because the efficiency of the transport system for chloride, where
~30 moles are transported for every mole oxygen
consumed, suggested that 2 chlorides are transported for every sodium pumped out by Na-K-ATPase (Silva et al., ’80). This stoichiometry was
confirmed by kinetic studies in isolated perfused
rectal gland tubules (Greger and Schlatter, ’84)
and in the isolated perfused rectal gland (Silva
and Myers, ’86). The cotransporter is activated
when the secretion of chloride is stimulated. Activation of isolated perfused rectal glands or isolated rectal gland tubules stimulated both the
secretion of salt and the binding of [3H]benzmetanide, suggesting that the Na-K-Cl cotransport system is activated as part of the process of
stimulation of secretion (Forbush et al., ’92; Lytle
and Forbush, ’92). The activation of the cotransporter is the result of cAMP dependent phosphorylation at serine and threonine residues (Lytle
and Forbush, ’92; Torchia et al., ’92), and appears
to be triggered by a fall in intracellular chloride
(Lytle and Forbush, ’92).
POTASSIUM CONDUCTANCE
The model for the secretion of chloride predicted
that potassium recirculates across the basolateral
membrane because the concentration of potassium
in the secretion of the gland was very small (Silva
et al., ’77). The importance of the recirculation of
potassium across the basolateral membrane was
evidenced by studies in which barium chloride in
the perfusate inhibited the secretion of chloride.
The effect of barium chloride was dose-dependent
and reversible (Silva et al., ’81). The effect of
barium was thought to be the result of depolarization of the rectal gland cell, a fact that was
confirmed by the observations of Greger et al. in
isolated perfused rectal gland tubules (Greger et
al., ’87; Greger and Schlatter, ’84).
CHLORIDE CONDUCTANCE
The model also predicted that a chloride channel facilitated the diffusion of chloride across the
506
P. SILVA ET AL.
luminal membrane. Indeed, stimulation of the secretion of chloride is associated with a fall in intracellular concentration of chloride both in
isolated perfused rectal glands and isolated rectal gland cells in culture, suggesting an increase
in the luminal permeability for chloride (Lear et
al., ’92; Silva et al., ’79). It should be noted that
the measurement of intracellular chloride concentration using chloride selective microelectrodes
showed no change in intracellular chloride activity at a time when chloride secretion was enhanced by theophylline and 8-bromo-cAMP (Welsh
et al., ’83). However, this observation suggests that
stimulation is associated with an enhancement of
net Cl movement across both the apical and
basolateral membranes. In a series of experiments
in isolated perfused rectal gland tubules Greger
et al., were able to directly demonstrate the presence of cyclic AMP stimulated chloride conductances in the apical membrane of rectal gland cell
(Gogelein et al., ’87; Greger et al., ’85, ’87). This
cyclic AMP dependent conductance has also been
demonstrated in cultured rectal gland cells (Devor
et al.,’95; Moran and Valentich, ’91; ’93). Furthermore, injection of rectal gland mRNA into frog oocytes results in the expression of a cyclic AMP
stimulated chloride conductance (Sullivan et al.,
’91). Thus, chloride leaves the rectal gland cell via
a chloride conductance present in the apical membrane in response to cyclic AMP. This conductance
has recently been cloned and found to be 75% homologous with the CFTR found in the apical membrane of human respiratory epithelia (Marshall
et al., ’91).
CURRENT MODEL FOR CHLORIDE
TRANSPORT BY THE RECTAL GLAND
Figure 1 shows the current model for the secretion of chloride by the rectal gland. Chloride enters the cell across the basolateral cell membrane
via the sodium: potassium: 2 chloride cotransporter. The energy for this electroneutral uptake
step is provided by the gradient for sodium directed into the cell. The gradient for sodium is
maintained by Na-K-ATPase present also in the
basolateral cell membrane. Also present in the
basolateral cell membrane is a potassium conductance that recirculates the potassium. Chloride
leaves the cell across the luminal membrane via
the chloride conductance CFTR. Thus, the transport of chloride by the rectal gland is similar to
that of other chloride transporting cells present
in vertebrate organisms.
Fig. 1. Model for the secretion of chloride in the rectal
gland. Chloride enters the cell across the basolateral cell membrane via the sodium: potassium: 2 chloride cotransporter.
The energy for this electroneutral uptake step is provided by
the gradient for sodium directed into the cell. The gradient
for sodium is maintained by Na-K-ATPase present also in
the basolateral cell membrane. Also present in the basolateral
cell membrane is a potassium conductance that recirculates
the potassium. Chloride leaves the cell across the luminal
membrane via the chloride conductance CFTR. Adenylate cyclase activates the secretion of chloride by activating the apical chloride conductance, CFTR, the sodium: potassium 2
chloride cotransporter and possibly Na, K, ATPase.
CONCLUSIONS
The rectal gland has been a powerful tool in
the elucidation of the different steps involved
in the transepithelial transport of chloride. The
model described for the transport of chloride in
the rectal gland has been succesfully applied
to all other furosemide-sensitive chloride-transporting epithelia. The characteristics of the
transport of chloride by the rectal gland are in
fact representative of those of transporting epithelia throughout the vertebrates, from the fish
gill to the mammalian cornea. Many of fundamental steps in the transport of chloride were
first described in this gland, to name a few remarkable observations: Na-K-ATPase purified
from this gland was used to link for the first
time the biochemical activity of the enzyme with
its transport properties; the sodium: potassium:
2 chloride cotransporter was first described in
membrane vesicles from this gland; the sodium:
RECTAL GLAND TRANSPORT MECHANISMS
potassium: 2 chloride cotransporter was first
cloned from this gland.
ACKNOWLEDGMENTS
All work conducted in the authors laboratory
was supported by grants from the USPHS NIH
NIDDK, NIEHS; NSF; and the American Heart
Association: Maine Affiliate. The authors acknowledge the help of all the dedicated assistants and
of the many colleagues that made these studies
possible.
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