-Adrenergic receptors regulate human lymphocyte
amiloride-sensitive sodium channels
James K.
Bubien,
Trudy
Cornwell,
Anne Lynn
Bradford,
Catherine
M.
Fuller,
Michael D.
DuVall, and
Dale J.
Benos
Departments of Physiology and Biophysics and Pathology,
University of Alabama at Birmingham, Birmingham, Alabama 35294
 |
ABSTRACT |
Two independent
signal transduction pathways regulate lymphocyte amiloride-sensitive
sodium channels (ASSCs), one utilizing cAMP as a second messenger and
the other utilizing a GTP-binding protein. This implies that two plasma
membrane receptors play a role in the regulation of lymphocyte ASSCs.
In this study, we tested the hypothesis that
1- and
2-adrenergic receptors
independently regulate lymphocyte ASSCs via the two previously
identified second messengers. Direct measurements indicated that
norepinephrine increased lymphocyte cAMP and activated ASSCs. The
2-specific inhibitor,
yohimbine, blocked this activation, thereby linking
2-adrenergic receptors to ASSC
regulation via cAMP. The
1-specific ligand, terazosin,
acted as an agonist and activated lymphocyte ASSCs but inhibited ASSC
current that had been preactivated by norepinephrine or
8-(4-chlorophenylthio) (CPT)-cAMP. Terazosin had no effect on the
lymphocyte whole cell ASSC currents preactivated by treatment with
pertussis toxin. This finding indirectly links
1-adrenergic receptors to
lymphocyte ASSC regulation via GTP-binding proteins. Terazosin had no
direct inhibitory or stimulatory effects on
,
,
-endothelial
sodium channels reconstituted into planar lipid bilayers and expressed
in Xenopus oocytes, ruling out a direct interaction between terazosin and the channels. These findings support the hypothesis that both
1- and
2-adrenergic receptors independently regulate lymphocyte ASSCs via GTP-binding proteins and
cAMP, respectively.
norepinephrine; amiloride; lymphocytes; sodium channel
 |
INTRODUCTION |
PRAZOSIN, TERAZOSIN, AND doxazosin are members of a
class of compounds that specifically interact with
1-adrenergic receptors. These
drugs have some efficacy as antihypertensive agents. In a random study,
these drugs were found to control blood pressure in 43% (106/245) of
individuals with mild to moderate hypertension (7). Unlike nonspecific
-receptor antagonists such as phentolamine, or direct vasodilators
such as Ca2+ channel inhibitors,
1-specific compounds do not
produce significant reflex tachycardia (10, 12, 13). Also, these
compounds have been shown to be reasonably safe for long-term
antihypertensive therapy either alone or in combination with other
antihypertensive agents (11).
It has long been known that
-adrenergic receptor antagonists were
therapeutically effective in disorders of bladder emptying. (16).
Recently,
1-specific inhibitors
have been approved for use in remitting some of the symptoms of benign
prostrate hyperplasia (9). Thus these drugs are now being taken by
normotensive and hypertensive individuals.
The most significant side effects of
anti-
1-receptor therapy are
orthostatic hypotension, which tends to remit with time, peripheral
edema, increased urination, and modest weight gains (6). All of these
effects have been attributed to specific blockade of
1-receptors. However, little
direct evidence confirms this hypothesis or rules out the possibility
of other mechanisms of action for the class of
1-specific antagonists.
During studies to identify mechanisms that regulate amiloride-sensitive
Na+ channels expressed by human
peripheral blood lymphocytes, it was observed that
1-receptor antagonists
specifically inhibited amiloride-sensitive
Na+ currents exogenously
stimulated by norepinephrine. However, these drugs also inhibited the
channels when they were stimulated by membrane-permeant analogs of
cAMP. This treatment circumvents any plasma membrane receptors. Thus it
was hypothesized that, in addition to blockade of
1-receptors, these compounds
may directly inhibit amiloride-sensitive
Na+ channels, thereby acting as
K+-sparing diuretics. This
hypothesis was tested using a variety of electrophysiological and
molecular biological techniques. It was found that these compounds do
not directly inhibit the channels but, rather, regulate
amiloride-sensitive Na+ channels
via a complex interaction with plasma membrane receptors and second
messenger signal transduction pathways. These findings help to explain
some of the side effects of these compounds and provide direct evidence
for the mechanism underlying the specific antihypertensive efficacy in
a subset of individuals.
 |
METHODS |
Whole cell patch clamp.
Micropipettes were constructed using a Narashigi pp-83 two-stage
micropipette puller. The tips of these pipettes had an inner diameter
of ~0.3-0.5 µm and an outer diameter of 0.7-0.9 µm.
When filled with an electrolyte solution containing 100 mM potassium gluconate, 30 mM KCl, 10 mM NaCl, 20 mM HEPES, 0.5 mM EGTA, <10 nM
free Ca2+, and 4 mM ATP, at a pH
of 7.2, the electrical resistance of the tip was 1-3 M
. The
bath solution was serum-free RPMI 1640 cell culture medium. The
solutions accurately approximate the constitutive ionic gradients
across the cell membrane. Pipettes were mounted in a holder and
connected to the headstage of an Axon 200A patch-clamp amplifier
affixed to a three-dimensional micromanipulator system attached to the
microscope. The pipettes were abutted to the cells, and slight suction
was applied. Seal resistance was continuously monitored (Nicolet model
300 oscilloscope) using 0.1-mV electrical pulses from an electrical
pulse generator. After formation of seals with resistances in excess of
1 G
, another suction pulse was applied to form the whole cell
configuration by rupturing the membrane within the seal but leaving the
seal intact. Successful completion of this procedure was known when
capacitance suddenly increased but seal resistance did not change. The
magnitude of the capacitance increase is a direct function of the
membrane available to be voltage clamped (i.e., cell size). Typically, this capacitance is between 5 and 10 pF for activated peripheral blood
lymphocytes.
Once the whole cell configuration was obtained, the pipette solution
and the cellular interior equilibrated within 30 s. The cells were then
held at a membrane potential of
60 mV and clamped sequentially
for 800 ms each to membrane potentials of
160,
140,
120,
100,
80,
60,
40,
20, 0, 20, and 40 mV, returning to the holding potential of
60 mV for 1 s between each test voltage. This procedure induced inward
Na+ (at more hyperpolarized
potentials) and outward K+ (at
more depolarized potentials) currents to flow across the membrane. The
currents were recorded digitally and filed in real time. The entire
procedure was performed using a DOS Pentium computer modified for
analog-to-digital signals with pCLAMP 6 software (Axon Instruments,
Sunnyvale, CA).
Lymphocyte amiloride-sensitive
Na+ channel
purification.
Epstein-Barr virus (EBV)-transformed lymphocytes were grown in
continuous culture in RPMI 1640 + 10% fetal bovine serum at 37°C
and 5% CO2 (95% air) in
75-cm2 tissue culture flasks (1.4 × 107 cells/flask). Eight
flasks of cells were collected by centrifugation at 500 g for 5 min at 4°C. The
lymphocytes were washed twice with phosphate-buffered saline and
pelleted. The cell pellet was resuspended in a Dounce buffer (10 mM
Tris-Cl, pH 7.6, 0.5 mM MgCl2,
supplemented with 2 µg/ml DNase, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl
fluoride). The suspension was homogenized with 30 strokes in a Dounce
homogenizer. Tonicity restoration buffer (10 mM Tris-Cl, pH 7.6, 0.5 mM
MgCl2, and 600 mM NaCl) was added
to a final NaCl concentration of 150 mM. The homogenate was then
centrifuged at 500 g for 5 min, and the pellet was discarded. Then, 0.5 M EDTA, pH 8.0, was added to the
supernatant to a final concentration of 5 mM. This was centrifuged at
100,000 g for 45 min. The final pellet
was solubilized overnight in a solution of 10 mM
NaH2PO4,
pH 7.4, 10 mM
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10% glycerol, and protease inhibitors as above (no DNase).
For affinity purification, 5 mg of a polyclonal antibody against a
bovine kidney papillary Na+
channel protein complex were irreversibly bound to protein A immobilized on agarose gel (Pierce). Two milliliters of gel were packed
in a column and equilibrated with 50 mM sodium borate, pH 8.2. The gel
was resuspended in a solution of the IgG and incubated at room
temperature for 30 min. Nonbound antibody was washed from the gel with
borate buffer. Bound IgG was cross-linked to the protein A with a 6.6 mg/ml solution of dimethyl pimelimidate (Pierce) for 1 h at room
temperature. Unreacted imidate groups were blocked with 0.1 M
ethanolamine, pH 8.2, for 10 min at room temperature. Finally, the
column was washed with 0.1 M glycine, pH 2.8, and then borate buffer.
The column was equilibrated with 10 mM sodium phosphate, pH 7.5.
Solubilized lymphocyte membrane proteins were applied to the column,
allowed to run into the gel bed, and incubated there for 1 h at room
temperature. The column was drained of the protein solution and washed
with 10 mM sodium phosphate until the absorbance [optical density
at 280 nm (OD280)] of the
wash from the column was the same as that of phosphate buffer alone.
Bound protein was then eluted with 0.1 M glycine-HCl, pH 2.8, in
500-µl fractions that were neutralized with 1 M Tris, pH 8.0. Absorbance measurements at OD280
were used to determine which fractions contained antigen.
Immunopurified protein was subsequently reconstituted into
proteoliposomes and incorporated into planar lipid bilayers for electrophysiological examination.
,
,
-Endothelial
Na+ channel in
vitro transcription.
cDNAs for
-,
,- and
-rat endothelial
Na+ channels (rENaCs) in the
pSPORT vector (a gift of Dr. B. C. Rossier, University of Lausanne,
Switzerland) were transcribed as previously described (8). Briefly, the
respective vectors were linearized with
Not I and transcribed with T7
polymerase (Message Machine, Ambion). The integrity of the resulting
cRNAs was verified by denaturing 1% agarose-formaldehyde gel
electrophoresis. The RNA was diluted in RNase-free water to a
concentration of 500 ng/µl, and the three subunits were mixed in a
1:1:1 ratio, giving a final concentration for each subunit of 8.33 ng/50 nl. Oocytes were injected and then examined
electrophysiologically 48-h postinjection.
 |
RESULTS |
Receptor-mediated activation of the human lymphocyte
amiloride-sensitive Na+
channels.
We have shown previously that human lymphocytes express an
amiloride-sensitive Na+
conductance (5) that is regulated in a complex manner involving both
protein kinase A (PKA)-mediated phosphorylation and a pertussis toxin-sensitive GTP-binding protein (4). These findings suggested that
plasma membrane receptors may play a role in the regulation of the
conductance. To test this hypothesis, EBV-transformed human lymphocytes
were whole cell patch clamped and exposed to receptor-specific agonists. Figure 1,
top, shows that
norepinephrine induced a specific activation of the inward conductance.
However, as shown in Fig. 1, bottom,
the
-adrenergic agonist, isoproterenol, had no effect on the whole
cell currents. Thus
-adrenergic receptors must mediate this agonist
effect of norepinephrine.

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Fig. 1.
Whole cell current records from Epstein-Barr virus (EBV)-transformed
human lymphocytes. These records are representative of 3 repetitions.
Top: in the absence of exogenous
stimulation, human lymphocytes conduct only minimal
Na+ (downward deflections,
top left). Superfusion with
norepinephrine induces a specific activation of inward
Na+ conductance
(top right).
Bottom: -adrenergic-specific
agonist (isoproterenol) had no effect on whole cell clamped lymphocyte
currents. Thus agonist effect of norepinephrine was most likely
mediated via -adrenergic receptors.
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Figure 2 shows the dose efficacy relation
for norepinephrine-mediated amiloride-sensitive
Na+ channel activation. The
agonist effect of norepinephrine was completely prevented by
pretreatment with the
2-specific inhibitor yohimbine
(300 nM, n = 3) and also completely
reversed by superfusion with the cAMP-specific antagonist
Rp-CPT-cAMP (100 µM). Direct measurement confirmed that norepinephrine acutely increased cellular cAMP in a time frame consistent with that required for
norepinephrine-mediated activation of the amiloride-sensitive
Na+ conductance (i.e., <5 min).
Table 1 shows that cellular cAMP concentrations increased more than an order of magnitude in response to
exposure to 30 µM norepinephrine and reached a maximum (presumably limited by substrate) within 5 min. These findings suggest that the
agonist effect of norepinephrine on lymphocyte amiloride-sensitive Na+ channels was mediated by
2-adrenergic receptors coupled
to cAMP-mediated signal transduction via adenylyl cyclase. However, the
agonist effect of norepinephrine and direct activation of cAMP-mediated signal transduction with 40 µM CPT-cAMP could also be completely reversed by the
1-specific
antagonists prazosin, doxazosin, and terazosin (Fig.
3), with an
IC50 of ~30 nM for all three
drugs. Figure 4 shows the mean dose
efficacy relation for terazosin. This inhibition could not be mediated
by receptor blockade because channel activation by cAMP is downstream
of the agonist receptor interaction as well as the production of
cellular cAMP by adenylyl cyclase. Also, cAMP-mediated channel
activation could not be inhibited by the potent
-adrenergic receptor
antagonist phentolamine at concentrations up to 1 µM. Figure
5 shows that phentolamine had no effect on
the whole cell currents activated in response to superfusion with 40 µM CPT-cAMP.

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Fig. 2.
Efficacy of norepinephrine was determined by measuring the chord
conductance between clamp potentials of 140 mV and the reversal
potentials, normalizing to the maximum conductance obtained for each
cell (n = 3), with each cell serving
as its own control. Apparent inhibition of the conductance at low
concentrations of norepinephrine (not statistically significant) may
have resulted from the simultaneous action of norepinephrine on
different classes of -adrenergic receptors.
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Fig. 3.
These whole cell clamp records are representative of at least 3 repetitions with each compound. They show identical experiments on
separate cells in which the inward conductance was stimulated with cAMP
and subsequently inhibited by 100 nM terazosin
(left) and doxazosin
(right). Prazosin (not shown) also
inhibited the lymphocyte Na+
conductance. Thus the effect appears to be a class effect and not
related only to specific class members.
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Fig. 4.
Mean dose-efficacy relation for terazosin-mediated inhibition of
cAMP-activated amiloride-sensitive
Na+ channel current in lymphocytes
(n = 4).
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Fig. 5.
These whole cell current records from an EBV-transformed human
lymphocyte show that there is very little basal whole cell inward
current (top left). However, cAMP
specifically increases the inward current (top
right), and -adrenergic antagonist (phentolamine)
has no effect on the activated current (bottom
left). Bottom right
shows that the currents return to the basal state once the test
compounds are washed out. Experiment is representative of 3 repetitions.
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Phentolamine has a different structure than the
1-specific compounds and does
not have the inhibitory effect on the cAMP-activated Na+ conductance. However,
phentolamine is a potent
-adrenergic receptor antagonist that
inhibits both
1- and
2-adrenergic receptors (1).
Because phentolamine had no effect on the ability of cAMP to activate
the conductance, the inhibitory effect of the
1-specific compounds was not
due to a general inhibition of
-receptors but rather an effect
specific for, and restricted to, the single class of
1-adrenergic receptor
antagonists.
1-Adrenergic receptor
blockers do not directly inhibit amiloride-sensitive
Na+ channels.
The lymphocyte whole cell current findings led to the hypothesis that
the
1-specific compounds may
have directly inhibited lymphocyte amiloride-sensitive
Na+ channels. This hypothesis was
tested by examining the effects of terazosin on amiloride-sensitive
Na+ channels purified from human
lymphocytes and incorporated into planar lipid bilayers and
,
,
-rENaC-specific amiloride-sensitive currents expressed in
Xenopus oocytes. Figure
6 shows the results of these experiments.
In both the planar lipid bilayer and in voltage-clamped oocytes
expressing
,
,
-rENaC, terazosin failed to inhibit
single-channel activity and whole oocyte current, respectively. In the
same preparations, however, amiloride produced significant inhibition
of the currents. Thus the simple hypothesis that
1-specific antagonists directly
block amiloride-sensitive Na+
channels proved to be incorrect.

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Fig. 6.
Left: single-channel activity recorded
from Na+ channel complexes
purified from human lymphocytes. Middle
left shows that 1 µM terazosin (10 times the amount
required to completely block the cAMP-activated
Na+ conductance in whole cell
clamped lymphocytes) had no effect on the single-channel activity or
conductance, whereas 10 µM amiloride completely inhibited the
activity. Right: whole cell current
records from a Xenopus oocyte injected
with , , -rat endothelial
Na+ channel (rENaC) subunits. As
in the bilayer, a high concentration of terazosin had no effect on the
current, but amiloride inhibited more than 50% of the whole cell
current in the same oocyte.
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|
Terazosin activates lymphocyte amiloride-sensitive
Na+ channels.
Because terazosin failed to directly block the amiloride-sensitive
Na+ channels in bilayer and oocyte
preparations, it was hypothesized that its action on
amiloride-sensitive Na+ channels
was mediated via a receptor mechanism that was not present in the
reconstituted experimental systems. Therefore, EBV-transformed human
lymphocytes were whole cell clamped and superfused with 100 nM
terazosin. In the absence of a prior stimulus, terazosin itself acted
as an agonist and induced specifically the activation of the
amiloride-sensitive Na+
conductance. A typical experiment is shown in Fig.
7, right. The finding that terazosin activated lymphocyte amiloride-sensitive Na+ channels independently
confirmed, in a third system, that the drug did not directly block
previously activated channels. Also, these findings ruled out the
possibility that the signal transduction mechanism for
terazosin-mediated amiloride-sensitive
Na+ channel activation involved
cAMP because, when 40 µM CPT-cAMP was added to the superfusate, the
inward terazosin-activated amiloride-sensitive Na+ conductance was completely
inactivated (Fig. 7, bottom right). This maneuver is opposite in sequence to that shown in Fig. 3 in which
terazosin and doxazosin inhibited the amiloride-sensitive Na+ conductance that had been
previously activated by treatment with 40 µM CPT-cAMP. No such
effects were observed when the
2-adrenergic receptor-specific
antagonist, yohimbine, was superfused (Fig. 7,
left). Thus yohimbine completely
prevented the agonist action of norepinephrine but had no activating
effect itself. Also, yohimbine did not interfere with the activation of
the inward conductance by CPT-cAMP. These findings demonstrate directly
that yohimbine is a passive receptor antagonist in this model system.
The differential effects of terazosin and yohimbine demonstrate
directly that two independent signal transduction pathways downstream
from both
1- and
2-adrenergic receptors converge
on a single effector mechanism (in this case, amiloride-sensitive
Na+ channels) to regulate its
activity.

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Fig. 7.
Representative whole cell clamp current records from human lymphocytes
(n = 4) showing that the
2-specific antagonist yohimbine
has no agonistic effects on lymphocyte plasma membrane conductance and
does not inhibit activation of the amiloride-sensitive
Na+ channels by
8-(4-chlorophenylthio) (CPT)-cAMP
(left). In contrast, superfusion
with the 1-specific
"antagonist," terazosin, induces specific activation of the
amiloride-sensitive inward Na+
conductance, which can be subsequently inhibited by the addition of
CPT-cAMP to the superfusate (right).
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Phentolamine prevents terazosin-mediated activation of lymphocyte
amiloride-sensitive
Na+ channels.
Phentolamine is an
-adrenergic receptor antagonist with a different
chemical structure unrelated to the class of
1-specific drugs that does not
have any direct effect on lymphocyte amiloride-sensitive Na+ channels (Fig.
8, top
right) or prevent cAMP-mediated activation of
lymphocyte amiloride-sensitive Na+
channels (Fig. 5). However, phentolamine prevents completely the
agonist effect of terazosin (Fig. 8, bottom
left), in a reversible manner. After the phentolamine
was washed out, the agonist effect of terazosin was readily apparent
(Fig. 8, bottom right). These findings add further support to the hypothesis that the regulatory effects (i.e., agonist and apparent antagonist) of terazosin on lymphocyte amiloride-sensitive Na+
channels are transduced via
1-adrenergic receptors.

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Fig. 8.
Whole cell currents from a single human lymphocyte. Identical outcomes
were observed in 3 additional cells. Generic -adrenergic receptor
antagonist, phentolamine, had no independent effect on human lymphocyte
whole cell currents (top right). In
the presence of phentolamine, terazosin failed to alter the whole cell
currents (bottom left), suggesting
that phentolamine prevented the activating effect by -receptor
inhibition. When the phentolamine was washed out, activating effect of
terazosin was readily apparent (bottom
right).
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Inhibition of
2-adrenergic receptors does
not inhibit the agonist effect of terazosin.
Nonspecific blockade of
-adrenergic receptors with phentolamine was
sufficient to prevent the activating effect of terazosin. To rule out
the possibility that
2-receptor
inhibition played a role in the phentolamine-mediated antagonism, the
experiments were repeated using the
2-specific inhibitor, yohimbine
(300 nM). Unlike terazosin, this inhibitor had no endogenous effect on
lymphocyte whole cell conductance (Fig. 9,
top right). Also, yohimbine
pretreatment failed completely to antagonize the agonist effect of
terazosin (Fig. 9, bottom left).
However, the terazosin-activated inward
Na+ currents were completely
inhibited by 2 µM amiloride (Fig. 9, bottom
right). These findings confirm that the regulatory
effects of terazosin on lymphocyte amiloride-sensitive
Na+ channels are independent of
2-adrenergic receptors. The
findings are consistent with the
1 specificity of terazosin and
support the hypothesis that the agonist and apparent antagonist effects of terazosin are both mediated via
1-adrenergic receptors. The mean current-voltage relations for these experiments are summarized in
Fig. 10. There are no significant
differences between the activated current records, regardless of
treatment. Likewise, there are no significant differences between the
mean basal current voltage relations and the current-voltage relations
when the currents are inactivated by treatment. As a means of
comparison, the conductance for each treatment was calculated from the
mean whole cell current measured at a membrane potential of
140
mV. The conductance was calculated as the chord between the mean
current amplitude (in pA/10 pS) and the reversal potential for each
cell studied. The average results are given in Table
2.

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Fig. 9.
Whole cell clamped human lymphocytes were superfused initially with the
2-specific antagonist,
yohimbine, which had no independent effect on the currents. Experiment
was repeated 4 times with identical results. This compound did
completely inhibit the agonistic effect of norepinephrine. In the
continued presence of yohimbine, terazosin added to the superfusate
specifically activated the inward currents. Activated current was
subsequently inhibited by the addition of amiloride to the
superfusate.
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Fig. 10.
These current-voltage relations show the distinct activation and
inactivation of the lymphocyte amiloride-sensitive whole cell
Na+ conductance.
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 |
DISCUSSION |
We have previously shown that CPT-cAMP and pertussis toxin regulate
amiloride-sensitive Na+ channels
expressed by human lymphocytes (4) and that when applied sequentially
these compounds activate and subsequently inactivate these channels.
CPT-cAMP is an active membrane-permeant analog of cAMP that activates a
second messenger cascade resulting in PKA-mediated phosphorylation of
susceptible cellular proteins. Pertussis toxin-ADP ribosylates
susceptible GTP-binding proteins. Both agents affect cellular second
messenger signaling cascades. These signaling cascades usually link
plasma membrane receptors to a variety of cellular effectors. Thus the
findings demonstrate directly that lymphocytes express plasma membrane
receptors capable of regulating amiloride-sensitive
Na+ channel function, given the
proper stimulus. In this report, we demonstrate directly that
-adrenergic receptors regulate amiloride-sensitive Na+ channel function in human
lymphocytes.
Model of adrenergic receptor regulation of lymphocyte
amiloride-sensitive
Na+ channels.
The experimental findings reveal a set of agonist and antagonist
effects between compounds that interact with
-adrenergic receptors
and a single effector set (i.e., amiloride-sensitive Na+ channels). The findings
demonstrate the complexity with which these channels can be regulated
and manipulated. To better understand this complexity, a model
summarizing the findings is shown schematically in Fig.
11. Because norepinephrine, but not
isoproterenol, activates the lymphocyte amiloride-sensitive
Na+ conductance, the
receptor-channel interaction is restricted to
-adrenergic receptors,
ruling out
-adrenergic receptors. Also, by direct measurement, it
was found that norepinephrine increased lymphocyte cAMP. Additional
experiments showed that the agonist effect of norepinephrine was
completely reversed by the cAMP-specific antagonist
Rp-CPT-cAMP, thereby establishing the
second messenger pathway linking the norepinephrine-receptor
interaction specifically to the cAMP-mediated signal transduction
pathway. Also, yohimbine at 300 nM (the concentration used in
these experiments) is a specific
2-antagonist. Thus inhibition
of the agonist effect of norepinephrine by 300 nM yohimbine supports
the conclusion that norepinephrine specifically increases lymphocyte
amiloride-sensitive Na+
conductance via
2-adrenergic
receptors, coupled to cAMP-mediated signal transduction.

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Fig. 11.
Schematic representation of the receptors and signal transduction
pathways that regulate the amiloride-sensitive
Na+ conductance of human
lymphocytes. PKA, protein kinase A.
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|
Terazosin is a ligand that interacts specifically with
1-adrenergic receptors.
Terazosin also has a specific agonistic effect on the lymphocyte
amiloride-sensitive Na+
conductance. The effect is not antagonized by yohimbine, ruling out
crossover with
2-receptors.
However, the effect is antagonized by phentolamine, which blocks both
1- and
2-adrenergic receptors. The
effect is also reversed by CPT-cAMP, thereby ruling out cAMP-mediated signal transduction as the mechanism for terazosin-induced activation of the conductance. The signal transduction pathway is not known but
may involve a pertussis toxin GTP-binding protein because pertussis
toxin has been shown to regulate the conductance in a reciprocal manner
with cAMP just as terazosin does (4). When pertussis toxin-treated
lymphocytes (with activated amiloride-sensitive Na+ channels) were treated with
100 nM terazosin, there was no inhibition of the inward conductance
(Table 2). If the hypothesis is that a GTP-binding protein
is involved in the
1-mediated
signal transduction signaling pathway, this is the expected result,
because pertussis toxin acts downstream from the receptor by directly
catalyzing the ADP ribosylation of certain GTP-binding proteins. Thus
the lack of inhibition by terazosin on pertussis toxin-activated
currents is consistent with the hypothesis that the
1-adrenergic receptor may
regulate lymphocyte amiloride-sensitive
Na+ channels by means of a
GTP-binding protein.
The physiological role of amiloride-sensitive
Na+ channels in human lymphocytes
is unknown. Traditionally, this type of channel has been linked to the
apical membrane of principal cells of the renal cortical collecting
duct. Whole cell experiments similar to those described in this report
have been performed on principal cells freshly isolated from rat
cortical collecting tubules with similar results (i.e., cAMP-mediated
specific activation of an amiloride-inhibitable inward current) (3). In
this setting, the role of the channels is as the rate-limiting step in
the reabsorption of salt and water (15). Lymphocytes do not perform
this function. Likewise, the physiological role of adrenergic receptors
is to transduce signals from the nervous system to effector organs such as the heart (2). Peripheral blood lymphocytes do not ordinarily associate specifically with nerve terminals. Also, the effective norepinephrine concentration in these experiments far exceeded that
normally found in plasma (0.3 µM) (2). Thus there is no established
physiological role for these receptors within the context of the
"normal" physiological functions of peripheral blood lymphocytes.
It is possible that expression may be vestigial. It is also possible
that there is a role for these receptors and channels, but the cellular
physiology is obscure. This lack of a physiological role or our
understanding of a role does not, however, undermine the regulatory
linkage between
-adrenergic receptors and amiloride-sensitive
Na+ channels demonstrated directly
by these experiments. Our current limited understanding of the
physiological role for Na+
channels and adrenergic receptors in lymphocytes does not diminish the
utility of this model for investigating and furthering our understanding of the mechanisms that regulate them. Also, it is possible that these same or similar regulatory pathways are also expressed by cells such as renal principal cells, where the
physiological role of amiloride-sensitive
Na+ channels is well established
(14).
One difference in the receptor-mediated regulation of
amiloride-sensitive channels expressed by renal principal cells and lymphocytes is that the peptide hormone vasopressin activates the whole
cell current (3) and increases Na+
flux in renal collecting ducts (14). There is no response to vasopressin by lymphocytes (not shown). It is known that the signal transduction pathway linking the vasopressin receptor to the principal cell amiloride-sensitive Na+
channels utilizes cAMP (14). Evidence was presented here showing that
the
2-adrenergic receptor
regulates lymphocyte amiloride-sensitive Na+ channels via cAMP. Thus there
is evidence for two distinct plasma membrane receptors that utilize the
same second messenger pathway to regulate the same effector (i.e.,
amiloride-sensitive Na+ channels)
in two different cell types.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the efforts of V. Shlyonsky, A. Maximov, and
B. Berdiev for their participation in obtaining the bilayer data.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants DK-52789 to J. K. Bubien and DK-37206 to D. J. Benos.
J. K. Bubien is an Established Investigator of the American Heart
Association.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. K. Bubien, Dept. of Physiology and
Biophysics, 876 McCallum Bldg., Univ. of Alabama at Birmingham,
Birmingham, AL 35294.
Received 25 February 1998; accepted in final form 2 June 1998.
 |
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