alpha -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
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha 1- and alpha 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 alpha 2-specific inhibitor, yohimbine, blocked this activation, thereby linking alpha 2-adrenergic receptors to ASSC regulation via cAMP. The alpha 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 alpha 1-adrenergic receptors to lymphocyte ASSC regulation via GTP-binding proteins. Terazosin had no direct inhibitory or stimulatory effects on alpha ,beta ,gamma -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 alpha 1- and alpha 2-adrenergic receptors independently regulate lymphocyte ASSCs via GTP-binding proteins and cAMP, respectively.

norepinephrine; amiloride; lymphocytes; sodium channel

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

PRAZOSIN, TERAZOSIN, AND doxazosin are members of a class of compounds that specifically interact with alpha 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 alpha -receptor antagonists such as phentolamine, or direct vasodilators such as Ca2+ channel inhibitors, alpha 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 alpha -adrenergic receptor antagonists were therapeutically effective in disorders of bladder emptying. (16). Recently, alpha 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-alpha 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 alpha 1-receptors. However, little direct evidence confirms this hypothesis or rules out the possibility of other mechanisms of action for the class of alpha 1-specific antagonists.

During studies to identify mechanisms that regulate amiloride-sensitive Na+ channels expressed by human peripheral blood lymphocytes, it was observed that alpha 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 alpha 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
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Abstract
Introduction
Methods
Results
Discussion
References

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 MOmega . 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 GOmega , 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.

alpha ,beta ,gamma -Endothelial Na+ channel in vitro transcription. cDNAs for alpha -, beta ,- and gamma -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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 beta -adrenergic agonist, isoproterenol, had no effect on the whole cell currents. Thus alpha -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: beta -adrenergic-specific agonist (isoproterenol) had no effect on whole cell clamped lymphocyte currents. Thus agonist effect of norepinephrine was most likely mediated via alpha -adrenergic receptors.

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 alpha 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 alpha 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 alpha 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 alpha -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 alpha -adrenergic receptors.

                              
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Table 1.   Lymphocyte cAMP concentration


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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 alpha -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.

Phentolamine has a different structure than the alpha 1-specific compounds and does not have the inhibitory effect on the cAMP-activated Na+ conductance. However, phentolamine is a potent alpha -adrenergic receptor antagonist that inhibits both alpha 1- and alpha 2-adrenergic receptors (1). Because phentolamine had no effect on the ability of cAMP to activate the conductance, the inhibitory effect of the alpha 1-specific compounds was not due to a general inhibition of alpha -receptors but rather an effect specific for, and restricted to, the single class of alpha 1-adrenergic receptor antagonists.

alpha 1-Adrenergic receptor blockers do not directly inhibit amiloride-sensitive Na+ channels. The lymphocyte whole cell current findings led to the hypothesis that the alpha 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 alpha ,beta ,gamma -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 alpha ,beta ,gamma -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 alpha 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 alpha ,beta ,gamma -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.

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 alpha 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 alpha 1- and alpha 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 alpha 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 alpha 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).

Phentolamine prevents terazosin-mediated activation of lymphocyte amiloride-sensitive Na+ channels. Phentolamine is an alpha -adrenergic receptor antagonist with a different chemical structure unrelated to the class of alpha 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 alpha 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 alpha -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 alpha -receptor inhibition. When the phentolamine was washed out, activating effect of terazosin was readily apparent (bottom right).

Inhibition of alpha 2-adrenergic receptors does not inhibit the agonist effect of terazosin. Nonspecific blockade of alpha -adrenergic receptors with phentolamine was sufficient to prevent the activating effect of terazosin. To rule out the possibility that alpha 2-receptor inhibition played a role in the phentolamine-mediated antagonism, the experiments were repeated using the alpha 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 alpha 2-adrenergic receptors. The findings are consistent with the alpha 1 specificity of terazosin and support the hypothesis that the agonist and apparent antagonist effects of terazosin are both mediated via alpha 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 alpha 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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Table 2.   Mean amiloride-sensitive Na+ channel conductance

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -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 alpha -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 alpha -adrenergic receptors, ruling out beta -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 alpha 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 alpha 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.

Terazosin is a ligand that interacts specifically with alpha 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 alpha 2-receptors. However, the effect is antagonized by phentolamine, which blocks both alpha 1- and alpha 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 alpha 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 alpha 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 alpha -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 alpha 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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(3):C702-C710
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