Effects of extracellular purines on ion transport across the integument of Hirudo medicinalis
Institut für Tierphysiologie der Justus-Liebig-Universität Giessen, Wartweg 95, D-35392 Giessen Germany
* Author for correspondence (e-mail: Mikael.K.Schnizler{at}physzool.bio.uni-giessen.de)
Accepted 29 May 2002
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Summary |
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Extracellular purines regulate transepithelial Cl- secretion and Na+ absorption. In a variety of tissues we tested ATP and adenosine for their effects on epithelial transport. Examination of integuments from pondwater- and high-salinity-adapted leeches revealed different sensitivities for these purines. Apical and basolateral application of ATP both stimulated transepithelial Na+ uptake and Iami. Adenosine upregulated non-Na+ currents and acted from the basolateral side only. Apical Ca2+-free conditions attenuated these effects of purines on transepithelial currents. Extracellular UTP had no effect on ion transport.
Key words: leech, Hirudo medicinalis, Na+ transport, invertebrate, amiloride, cyclic AMP, ATP, adenosine, UTP
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Introduction |
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In the last 40 years, vertebrate epithelia have been the preferred models
for investigating Na+ transport in tight epithelia. Regulation of
Na+ absorption occurs in several ways
(Garty and Palmer, 1997), the
major stimulus in vertebrates being the mineralocorticoid aldosterone. This
steroid hormone mediates long-term adaptation by influencing the expression of
regulatory factors at both transcriptional and translational levels. Peptides,
for example antidiuretic hormone (ADH), bind to G-protein-coupled receptors
and exert their regulatory effects within minutes. Invertebrates such as the
leech also possess these phylogenetically old antidiuretic hormones, but they
lack the mineralocorticoid system found in vertebrates
(Oumi et al., 1994
;
Proux et al., 1987
; Salzet et
al., 1993
,
1995
;
Satake et al., 1999
). To
investigate the processes underlying Na+ transport across tight
epithelia in invertebrates, we used the integument of the leech Hirudo
medicinalis for electrophysiological measurements
(Milde et al., 2001
;
Schnizler and Clauss, 1998
;
Weber et al., 1993
,
1995
).
The regulatory properties of vertebrate epithelial Na+ channels
have been subject to intensive study over the last 10 years, and regulation of
these epithelial Na+ channels occurs by modulation of channel
activity within the apical cell membranes as well as by control of the total
number in surface-expressed channels
(Garty and Palmer, 1997). High
extracellular Na+ concentrations induce Na+
self-inhibition, a mechanism that downregulates amiloride-sensitive apical
Na+ entry (Palmer et al.,
1998
; Turnheim,
1991
). Furthermore, in a variety of epithelia, downregulation of
apical Na+ conductances was observed when intracellular
Na+ concentration increased. This autoregulative Na+
feedback inhibition represents a short-term adaptive process, and current
Na+ feedback inhibition models postulate an intracellular, as yet
unidentified, Na+-sensing receptor that mediates, through
G-protein-coupled mechanisms, a ubiquitin-protein ligase (Nedd4)-dependent
endocytosis and degradation of the epithelial Na+ channels
(Ishibashi et al., 1999
;
Komwatana et al., 1998
). The
involvement of G proteins was not confirmed in all cases, however
(Hubner et al., 1999
).
Living in fresh water and only occasionally entering brackish water
(Herter, 1939), Hirudo
medicinalis cannot be expected to be a euryhaline organism. Nevertheless,
our previous studies showed that the leech integument reduces electrogenic
Na+ uptake in the presence of high apical Na+
concentrations. This short-term adaptive downregulation was maintained for
several hours (as long as the experiments lasted) and quickly became
inoperative after a return to low-[Na+] conditions
(Weber et al., 1995
). Adult
leeches survive in water of up to 16
salinity for several months
(Boroffka, 1968
). There, they
behave as hyperosmotic osmoconformers, with hyporegulated [Cl-] in
the blood but an accumulation of short-chain carboxylic acids
(Nieczaj and Zerbst-Boroffka,
1993
). In the initial phase (within 4 h) of acclimation, osmotic
loss of water and uptake of salt were the prominent passive events.
Concentrations of Na+, K+, Cl- and of organic
anions in the blood were greatly increased
(Nieczaj and Zerbst-Boroffka,
1993
). After a few days, extracellular volume was restored. Our
interest in investigating whether there is long-term acclimation of
transintegumental ion transport during extended exposures to high-saline
conditions arose from these findings. We exposed leeches for several days to a
high-salinity environment (200 mmol l-1 NaCl) to elicit an
osmoregulatory long-term adaptation of Na+ uptake; the annelids
appeared to cope with this excessive physiological stress. The plasma
Na+ concentration of Hirudo medicinalis is approximately
115 mmol l-1 (Zerbst-Boroffka
and Wenning, 1986
), and an unhindered influx of Na+
from the 200 mmol l-1 Na+ environment would therefore
change the physiological situation from absorption to excretion of excess
Na+.
For comparative purposes, dorsal segments of these high-salt-adapted and of pondwater-adapted integuments were dissected for Ussing chamber experiments. The preparations were voltage-clamped, and the initial electrophysiological properties determined to expose any differences attributable to high-salt-adaptation at the level of integumental Na+ conductances. We measured total transepithelial Na+ uptake and the amiloride-sensitive Na+ current.
Extracellular nucleotides serve as agonists for a variety of membrane
receptors. Besides ionotropic P2X-type receptors, which comprise intrinsic ion
channels, two other large families of G-protein-coupled purine receptors, the
P2Y- and the P1-receptors with a multitude of subtypes, have been
characterized and cloned (Ralevic and
Burnstock, 1998; Surprenant et
al., 1995
). Expression of these receptors has been detected in a
variety of epithelia in which they function in the para- and/or autocrine
control of transepithelial ion-transport processes
(Ralevic and Burnstock, 1998
).
ATP, for example, may be released from epithelial cells under physiological
conditions (Schwiebert, 1999
).
A reciprocal regulation, stimulation of Cl- secretion accompanied
by a reduction in Na+ absorption, by extracellular trinucleotides
may be a common and cytosolic Ca2+-dependent principle of
ion-transport control, e.g. in renal or pulmonary epithelia
(Cuffe et al., 2000
;
Hayslett et al., 1995
;
McCoy et al., 1993
).
Nucleotides can regulate NaCl transport via P2Y- and P2X-type
receptors (McCoy et al.,
1999
). The distribution of some epithelial purinoceptors is
restricted to either the apical or the basolateral membrane, and this accounts
for the `side-specific' regulatory effects of extracellular nucleotides
(Casavola et al., 1996
,
1997
). In the present study, we
evaluate the regulatory impact of intercellular nucleotide messengers on the
control of ion transport across the leech integument. For this purpose, we
measured the effects of adenosine, ATP and the pyrimidine UTP on
Na+ transport and also investigated whether the removal of apical
Ca2+, which is known greatly to stimulate transintegumental current
(Prusch and Otter, 1977
;
Weber et al., 1995
),
interferes with purinergic control. Examination of pondwater- and
high-salt-adapted integuments indicated different sensitivities to
extracellular nucleotides.
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Materials and methods |
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A ventral incision was made in the leech body wall, and the intestine was detached and muscular layers were carefully scraped off. The dorsal integument of the subclitellar region was fixed on a needle-spiked ring. The Ussing chamber had an aperture of 0.5 cm2. The edges of the tissue were sealed with silicone grease. During experiments, both compartments of the Ussing chamber were continuously perfused (apical, at approximately 7 ml min-1; basolateral, at approximately 3 ml min-1). All experiments were performed at room temperature (18-24°C). A preparation of the dorsal integument (subclitellar region) was mounted in an Ussing type chamber. After measurement of the initial potential, Vinit, the transepithelial potential, VT, was allowed to equilibrate and then clamped to 0 mV. The short-circuit current (Isc) was recorded continuously, and the transepithelial resistance (RT) was calculated from the effects of 20 mV pulses on Isc. The amiloride-sensitive current (Iami) was measured as the decrease in Isc in the apical presence of apical 100 µmol l-1 amiloride. INa, the transepithelial Na+ current, was when apical NaCl was substituted with equimolar tetramethylammonium chloride (TMA-Cl). Readdition of apical Na+ re-established the Isc.
Solutions and chemicals
The basolateral Ringer's solution contained (in mmol l-1): 115
NaCl, 4 KCl and 1.8 CaCl2. In the apical solution, KCl was replaced
with TMA-Cl. In Na+-free solutions, NaCl was substituted by
equimolar concentrations of TMA-Cl. Ca2+-free solutions contained
0.5 mmol l-1 EDTA. All solutions were buffered with 5 mmol
l-1 Hepes and adjusted to pH 7.4 with Tris (Trizma-base).
APW contained (in mmol l-1): 1 NaCl, 0.05 KCl, 0.4 CaCl2 and 0.2 NaHCO3, pH 7.4. HSW contained (in mmol l-1): 200 NaCl, 1 KCl, 0.4 CaCl2 and 0.2 NaHCO3, pH 7.4.
Adenosine, ATP, UTP, cyclic AMP [8-(4-chlorophenylthio)-cAMP, cpt-cAMP] and 3-isobutyl-1-methyl-xanthine (IBMX) were obtained from Sigma.
Electrical measurements
Ag/AgCl wires in 1 mol l-1 KCl served as current- or
voltage-measuring electrodes. For conductive connection to bathing
compartments, 1 mol l-1 KCl/agar bridges were used. During
measurements, the transepithelial potential was voltage-clamped to 0 mV
(voltage-clamp amplifier; Nagel, Munich, Germany). The short-circuit current
(Isc) was recorded continuously on a stripchart recorder
and on computer (Apple IIsi, MacLab interface, chart recorder program: Analog
Digital Instruments). RT was calculated according to Ohm's
law from changes in Isc during superimposed 20 mV
pulses.
Statistical analyses
Data are presented as means ± S.E.M. N is the number of
experiments and animals. Statistical analyses were performed using paired or
independent Student's t-tests (Microcal Origin).
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Results |
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Regulation of epithelial Na+ uptake via purinergic
receptors
We tested different concentrations of ATP for their effect on
transepithelial ion conductances of APW- and HSW-adapted integuments. ATP was
applied to the basolateral side at concentrations of 10 µmol l-1
to 10 mmol l-1 (Fig.
1). Maximal stimulation was observed in all preparations with 1
mmol l-1 ATP, giving a concentration required for 50 % stimulation
(EC50) of approximately 80 µmol l-1. Exposure to 1
mmol l-1 ATP did not affect Isc in APW-adapted
integuments (98.7±6.9 %; N=5)
(Fig. 2). In HSW-adapted
integuments ATP induced a twofold (214.7±42.1 %) increase in
Isc, which resulted from the activation of Na+
conductances (Fig. 2). This, in
turn, could be attributed to a more than threefold (364±103.9 %)
increase in the amiloride-sensitive current. In comparison, in APW-adapted
integuments, Iami increased only slightly (by
187.5±33 %) at the cost of other Na+ conductances (not
shown). The ATP-induced Iami was larger than the
subsequently determined net Na+ transport (INa;
Table 2). This paradoxical
finding may be explained by additional effects of apical Na+-free
conditions on, for example, Cl- conductances. Higher
concentrations, such as 10 mmol l-1 ATP, partially deactivated
Isc again. This downregulation phenomenon in the presence
of 10 mmol l-1 ATP was observed in all experiments (data not
shown). Apical application of ATP (1 mmol l-1) also greatly
stimulated Isc, a result that was not further
investigated.
|
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|
Under apical Ca2+-free conditions, ATP had no effect on Isc across APW-adapted integuments (112.8±6.8 %). In contrast, upregulated Isc across HSW-adapted integuments under Ca2+-free conditions responded to 1 mmol l-1 ATP with a significant increase to 136.3±6.4 %. ATP preferentially stimulated Na+ uptake and had very little effect on non-Na+ conductances. This effect of ATP on transepithelial Na+ transport in HSW-adapted integuments was largely due to an upregulation of Iami by a factor of 235.9±49.5 %, whereas Iami increased to 185.4±27.9 % in APW-adapted integuments (not shown).
Certain types of P2Y receptor selectively bind uridine nucleotides but not
adenosine nucleotides (Lazarowski and
Boucher, 2001; Ralevic and
Burnstock, 1998
). UTP (1 mmol l-1) was added either to
the apical or basolateral compartment and tested for its effect on macroscopic
transepithelial ion conductances across APW- and HSW- adapted integuments.
Apical treatment with 1 mmol l-1 UTP left Isc,
INa and its amiloride-sensitive component
Iami unaffected in both integuments (data not shown).
While Iami of APW-adapted integuments responded somewhat
inconsistently to basolateral UTP, the Isc was reduced to
87 % compared with the control value and total INa was not
significantly affected. Under apical Ca2+-free conditions, UTP had
no effect on Isc. In addition, the basolateral side of
HSW- adapted integuments was insensitive to 1 mmol l-1 UTP under
both normal and Ca2+-free conditions.
Regulation of Na+ transport by adenosine
/Pl-receptors
We examined the effects of extracellular adenosine in the apical and the
basolateral compartments. Isc and Na+ currents
across APW- and HSW-adapted integuments were insensitive to apical adenosine
(data not shown). While Isc, INa and
Iami in APW-adapted integuments were largely unaffected by
basolateral application of adenosine, the Isc of
HSW-adapted integuments increased in a concentration-dependent manner
(Fig. 3). A maximal, stable and
non-transient activation of Isc was observed in the
presence of 10 mmol l-1 adenosine (204±35 % of control
amplitude; N=5; EC50=0.17 mmol l-1). In
contrast to the effect of ATP described above, the net increase in
Isc was due to a stimulation of non-Na+
conductances (Fig. 4).
Interestingly, in HSW-adapted integuments, adenosine increased
Iami current to 188.1±18.7 % without changing total
INa (Fig.
4). Basolateral treatment with 1 mmol l-1 adenosine,
after upregulation of Na+ currents by Ca2+-free
conditions, had no significant effect on Isc or on the
component of Isc carried by Na+.
Iami, however was slightly reduced to 79.5±5.6 % in
APW-adapted integuments, whereas it increased in HSW-adapted integuments to
175.6±65.2 % in response to adenosine. The size of this effect varied
substantially, but it was observed in all preparations tested.
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Table 2 summarizes the currents and the effects of apical Ca2+-free conditions in the basolateral presence of 1 mmol l-1 ATP, UTP and adenosine. Transepithelial ion conductances have been reported to be regulated by extracellular adenosine and through cyclic AMP/protein kinase A (PKA) signalling mechanisms. We treated APW- and HSW-adapted integuments in the absence of apical Ca2+ with membrane-permeant 100 µmol l-1 cpt-cAMP in combination with 1 mmol l-1 IBMX (an inhibitor of phosphodiesterases). However, this effect of cyclic AMP was abolished under Ca2+-free conditions. Both INa and the non-Na+ current were unaltered by cyclic AMP (Fig. 5). The amiloride-sensitive part of INa did not change in APW-adapted (94±6 %) or HSW- adapted integuments (102±8 %). The apical epithelial Na+ channels were therefore not affected.
|
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Discussion |
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Pondwater presents the leech integument with the problem of absorbing ions
against considerable electrochemical gradients. The apical transport of
Na+ requires energy and may be driven by proton-motive forces from,
for example, H+ V- ATPases
(Ehrenfeld and Klein, 1997;
Nelson and Harvey, 1999
). In
high-salinity conditions the situation for overall ion regulation is reversed.
Extracellular 200 mmol l-1 Na+ exceeds plasma
concentrations (115-140 mmol l-1)
(Prusch and Otter, 1977
;
Zerbst-Boroffka and Wenning,
1986
) and should therefore result in increased levels of body
salts. Shutdown of apical Na+ conductances and extrusion of excess
salt by, for example, nephridia may then balance total Na+ uptake
(Zerbst-Boroffka and Wenning,
1986
). To determine when long-term (transcriptional and/or
translational) regulation of ion transport sets in, one would have to perform
a series of time-course experiments that specify time required for changes in
electrical properties of the leech integument. However, such an approach is
difficult because this epithelium shuts down ion transport in the presence of
high apical Na+ concentrations. Na+ uptake recovers soon
after a return to low-[Na+] conditions
(Weber et al., 1995
; M.
Schnizler, personal observations). The recovery of Na+ transport
after a shift from apical high-Na+ conditions to low-Na+
conditions is repeatably reproducible and the phenomenon remains unchanged
even after exposure to high-Na+ conditions for more than 5 h. We
therefore decided to extend the high-salt challenge over several days, by
which time long-term adaptive processes could be expected to have occurred
(Nieczaj and Zerbst-Boroffka,
1993
). Recent models attribute triggering of the autoregulative
feedback inhibition of Na+ transport in tight epithelia to an
elevation in intracellular [Na+]
(Palmer et al., 1998
;
Turnheim, 1991
). The
phenomenon is a short-term adaptive process that starts immediately and is
reproducible for several cycles of transfer from low to high salinity. There
is evidence for Na+-sensing receptors that mediate the endocytosis
of epithelial Na+ channels and their subsequent degradation
(Hubner et al., 1999
;
Ishibashi et al., 1999
;
Komwatana et al., 1998
). The
feedback inhibition is therefore due to a reduction in the total number of
surface-expressed channels. We expected the high-salt exposure of several days
not only drastically to reduce transcellular Na+ transport but also
to affect the number of new vesicle-stored channels available for apical
membrane insertion. Long-term adaptation to high-salt conditions reduced
VT and Isc
(Table 1). However, although
transepithelial Na+ uptake was downregulated, the
amiloride-sensitive transcellular Na+ current
(Iami) was not affected
(Table 1).
Effect of Ca2+ and purinergic signalling
High extracellular Na+ concentrations are generally believed to
elevate cytosolic Ca2+ concentrations which, in turn, inhibit the
amiloride-sensitive Na+ channels, either directly or by
protein-kinase-C-mediated mechanisms
(Garty and Palmer, 1997).
Despite persistent evidence for the importance of Ca2+ in
short-term Na+ feedback inhibition, their direct involvement could
not be verified (Abriel and Horisberger,
1999
). It is not clear whether the downregulation of transcellular
Na+ uptake by apical Ca2+ results from interactions with
elements in the apical membranes or by Ca2+ entering the cells and
increasing cytosolic Ca2+ concentrations
(Abriel and Horisberger, 1999
;
Palmer et al., 1998
;
Turnheim, 1991
). In addition
to a direct and possibly non-specific action upon Na+ channels or
regulatory factors, G-protein-coupled Ca2+-sensing mechanisms may
come into play (Komwatana et al.,
1998
). Surface expression of such Ca2+ receptors has
been reported for several tissues (Brown et
al., 1993
; Riccardi,
2000
), but their presence in the leech integument must first be
verified.
One objective of our study was to investigate whether longterm acclimation
to high-salt conditions affects this short-term regulation of transcellular
Na+ uptake and its sensitivity to apical Ca2+. Both
high-salt- and pondwater-adapted integument responded to the removal of apical
Ca2+ with an increased Isc to values of similar
magnitude, and this was mainly due to an upregulation of
Iami (Table
1). Thus, the amiloride-sensitive Na+ conductances are
partly inhibited by the extracellular presence of Ca2+. This
stimulating effect of Ca2+ removal in the leech skin may be
correlated with downregulated Na+ transport since only under apical
high Na+-conditions could Isc be stimulated by
the removal of apical Ca2+
(Weber et al., 1995).
Transcellular transport of Ca2+ occurs in a variety of
epithelia, and apical entry of Ca2+ greatly affects intracellular
Ca2+ concentrations (Hoenderop
et al., 2000). In vertebrates, for example, there are epithelial
Ca2+ channels that lose their selectivity and transport monovalent
cations in the absence of extracellular Ca2+
(Vassilev et al., 2001
). Total
removal of apical Ca2+ can be expected to reduce cytosolic
[Ca2+]. Basolateral electrogenic 3Na+/1Ca2+
exchange processes are considerably downregulated in this situation, and this
may explain upregulation of transcellular Na+ transport in
Ca2+-free conditions (Blaustein
and Lederer, 1999
; Hoenderop
et al., 2000
). Interestingly, the stimulating effect of a removal
of Ca2+ was greater in HSW-adapted than in APW-adapted integuments,
but the final upregulated currents from HSW-adapted integuments were equal to
those from APW-adapted integuments (Table
1). We do not know the extent to which vesicular translocation of
new channels contributed to the effect of Ca2+, but as a result of
long-term adaptation, one would expect a marked reduction in de novo
synthesis of channels rather than the formation of channel-containing membrane
vesicles. The rapid upregulation observed indicated in situ
activation of silent, but surface-expressed, Na+ channels.
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P2 receptors |
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The principal agonists for P2X receptors appear to be ATP and its
derivatives, but not UTP. Neither basolateral nor apical application of 1
mmoll-1 UTP had a dramatic effect on Isc or
INa in integuments adapted to both conditions
(Table 2), which suggests the
existence of P2X-mediated processes. Following activation by ATP, ligand-gated
P2X receptors open their intrinsic non-selective cation channel, allowing
Na+ and Ca2+ to pass from the extracellular fluid into
the cell. Cellular release of ATP therefore provides one mechanism for
controlling intracellular Ca2+ concentration and downstream
transduction processes (Bean,
1992; Dubyak and el-Moatassim,
1993
). In general, P2X- and P2Y-receptor-induced regulation of
transepithelial Na+ absorption and/or Cl- secretion is
mediated by an increase in cytosolic [Ca2+]
(Cuffe et al., 2000
;
Mall et al., 2000
;
McCoy et al., 1999
) as a
result of extracellular Ca2+ entering the cell via P2X
receptors or a P2Y-induced release of Ca2+ from intracellular
stores. However, the absence of Ca2+ from apical solution did not
prevent the stimulating effect of basolateral ATP on Na+
conductances in HSW-adapted integuments
(Table 2).
Iami was again upregulated, although the response was less
pronounced. A significant contribution of Ca2+ influx to the
transduction of purinergic stimulation of Iami cannot be
ruled out since, for example, the previously observed upregulation of
Isc or the total INa in HSW-adapted
integuments by ATP are reduced or in some cases even prevented after removal
of apical Ca2+. A further explanation may be that Ca2+
greatly prestimulates amiloride-sensitive Na+ conductances, leaving
fewer quiescent apical Na+ channels to be activated in situ by
further, i.e. purine-induced, mechanisms. Hydrolysis or the conversion of
exogenous nucleotides by ecto-nucleotidases
(Harden et al., 1997
) may be
one explanation for the requirement of concentrations as high as 1
mmoll-1 ATP to evoke the maximal effect.
At present, no agonists or antagonists are available that discriminate
effectively between the families and subtypes of P2X and P2Y receptors
(Norenberg and Illes, 2000;
Ralevic and Burnstock, 1998
;
von Kugelgen and Wetter, 2000
)
and little is known about invertebrate orthologues and their pharmacology.
Thus, it is difficult to characterize the basolateral leech ATP receptors and
to prove a correlation between their relative expression levels and
environmental salinity using northern blots or quantitative reverse
transcriptase/ polymerase chain reactions.
P1 receptors
Adenosine has been reported to have pleiotropic actions in epithelia
(McCoy et al., 1993;
Olah and Stiles, 1992
).
Na+ uptake by tight epithelia, e.g. in the medullary collecting
duct cells of the rat kidney, is controlled by extracellular adenosine
(Yagil, 1994
). In A6 cell
monolayers (cells from Xenopus laevis nephron), A1 receptors are
located on the apical surface and regulate Cl- secretion or
inhibition of transepithelial Na+ transport via
stimulation of phospholipase C activity. On the basolateral surface, there are
A2A receptors, which stimulate Na+ uptake (Casavola et
al., 1996
,
1997
). This positive effect is
believed to be due to upregulation of cyclic AMP production followed by
stimulation of Na+/H+ exchange activity. The restricted
distribution of receptors to the apical or basolateral membrane and the
distinct post-receptor mechanisms are both responsible for the dual-control
regulation by adenosine of transepithelial ion conductances.
In the leech integument, addition of adenosine to the apical compartment failed to have any effect on macroscopic Isc. Instead, we found that activation of basolateral adenosine receptors only stimulated Isc in HSW-adapted integuments and not in pondwater-adapted controls. Net Na+ transport was not upregulated, and we assume that Cl- conductances are targets for this effect of adenosine (Fig. 4). The slope of the current traces indicated that this activation of HSW-adapted integuments was a monophasic but rather slow process (Fig. 3). Interestingly, the amiloride-sensitive portion of INa was enlarged at the expense of other Na+ conductances. This effect was abolished under Ca2+-free conditions.
After ligand-binding by certain purinoceptors, the signal is propagated in
some cases by cyclic AMP/PKA-mediated pathways
(Ralevic and Burnstock, 1998).
Under normal conditions, membrane-permeant cyclic AMP activates
amiloride-sensitive currents across leech integuments
(Weber et al., 1995
). We
applied cpt-cAMP after the effect of the removal of Ca2+ had
concluded, but could detect no convincing effect of this compound on
transintegumental ion conductances, however
(Fig. 5).
Iami across APW-adapted integuments was slightly reduced
rather than stimulated, and Iami across HSW-adapted
integuments was unchanged. Interestingly, the overall Na+ current,
INa was unaltered by the addition of cpt-cAMP
(Fig. 5), possibly indicating
that apical Ca2+-free conditions had stimulated the Na+
conductances to their maximum. High concentrations of adenosine were required
to induce the maximal response, so mechanisms other than binding to
metabotropic receptors must be taken into consideration. The nucleosides may
have entered the cells and initiated further processes.
In conclusion, the leech integument provides a useful model for investigating the regulation of ion transport in an invertebrate tight epithelium. Adaptation of leeches to high-salinity conditions altered most of the initial electrophyiological properties of the integuments but left the amiloride-sensitive Na+ current unaffected. Absence of apical Ca2+ stimulated the bioelectrical activities in integuments adapted to high-salinity conditions and pondwater to a similar extent. Long-term exposure to high (200 mmoll-1) NaCl concentration conferred ATP-sensitivity to the Isc and the INa, although there was little effect on control integuments. Our findings provided no evidence that UTP has any relevance in the control of ion-transport processes across the leech integument. Adenosine upregulated transepithelial non-Na+ currents in HSW-integuments, while APW-adapted preparations were largely unaffected by this nucleoside. The surface expression of adenosine/P1 receptors was restricted to the basolateral membrane. These disparities in sensitivity to extracellular purines may be caused by differentially expressed receptors or regulatory factors during adaptation to high-salt conditions. Finally, one can argue that high-salt stress is a situation that a leech will never face under natural conditions. Nevertheless, exposure to such physiological challenges provides an opportunity for detecting variances of macroscopic variables, for instance transepithelial ion conductances, which may indicate assimilative processes in a whole-animal model and initiate more fine-tuned approaches in future projects.
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Acknowledgments |
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