ENaC plays a role in regulated antibody secretion by hybridomas

Zhen-Hong Zhou and James K. Bubien

Department of Physiology & Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hybridomas are fused immortal lymphocytes that typically secrete monoclonal antibodies to a known antigen. Hybridomas express two ionic conductances that have properties consistent with epithelial sodium channel (ENaC) and CFTR. Both ion channels are expressed by lymphocytes. Both of these channels are known to play a role in epithelial cell physiology. However, the physiological role of these channels in lymphocytes is unclear. We tested the hypothesis that ENaC plays a role in the process of regulated antibody secretion. We have been able to demonstrate that hybridomas can be provoked to acutely secrete monoclonal antibodies by a variety of agonists. Concurrently, we were able to show that these same agonists activate amiloride-sensitive sodium currents in whole cell clamped hybridomas. Inhibition of ENaC by amiloride inhibited the acute provoked antibody secretion, thereby linking ENaC to the process of acute antibody secretion. Interestingly, the concentration of amiloride necessary to completely inhibit the provoked secretion was approximately an order of magnitude higher than the concentration necessary to inhibit all of the transmembrane current. However, because amiloride is a weak base, the equilibrium concentration necessary to produce partial inhibition was precisely in accord with the Ki for amiloride and ENaC, indicating that the inhibition was intracellular.

epithelial sodium channel; hybridoma


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE EPITHELIAL SODIUM CHANNEL ENaC is a sodium channel that is inhibited by amiloride (7). One function of this sodium channel is the vectorial transport of sodium across the apical membrane of principal cells located in the collecting duct of the distal renal nephron (11). We have used electrophysiological, biochemical, molecular biological, and immunofluorescent methods to demonstrate directly that ENaC is also expressed by lymphocytes (5). Expression of this sodium channel by lymphocytes has been of considerable assistance in elucidating the basic functional abnormality responsible for Liddle's disease (3). Lymphocytes have also proved useful for the diagnosis of ENaC-induced refractory hypertension (6).

Lymphocyte ENaC is regulated by factors that increase cellular cAMP, such as norepinephrine (via the alpha 1-adrenergic receptor) (2). Aldosterone also acutely activates human lymphocyte ENaC (12). The pathway that transduces aldosterone-mediated ENaC activation utilizes a transmethylation reaction (12). We have also demonstrated a lymphocyte ENaC sensitivity to pertussis toxin-mediated ADP-ribosylation of G proteins (4). These studies of ENaC expressed by human lymphocytes have provided considerable new knowledge about cellular mechanism that influence the ion channel function of ENaC. They have not, however, addressed the question of the physiological function of ENaC in lymphocytes. Clearly, this sodium channel in peripheral blood lymphocytes cannot control vectorial salt and water movement as it does in polarized epithelia. Thus we hypothesized that ENaC may serve some other cellular function in lymphocytes.

Lymphocytes produce and secrete a variety of inflammatory mediators such as antibodies and cytokines. These products are not secreted in high levels constitutively; rather, they are secreted in response to infection or injury. Thus the secretion of these mediators must be responsive to external signals such as antigen binding or to other inflammatory mediators. Because we had determined previously that ENaC current increased significantly in response to agonists and that ENaC expression in the plasma membrane increased significantly in response to these same agonists (5), we hypothesized that secreted products would increase acutely in response to the same agonists. To test these hypotheses, we measured antibody secretion by hybridomas in response to agonists that increased ENaC current.

Very little is known concerning the precise mechanism whereby immunoglobulins are transported to the plasma membrane and secreted. Immunoferritin cytochemistry on ultrathin frozen sections of normal plasma cells and plasmacytoma cells revealed vacuoles on the trans-side of the Golgi complex containing IgG. It has been speculated that these structures were immunoglobulin secretory vacuoles (8). Another study showed that the polar accumulation of secretory immunoglobulin was contained within vesicles and that the polar multivesiculated structure was responsible for antibody release, either as a free form or packaged within satellite vesicles (10). The specific makeup of the vesicular membrane, specifically the identification of ion channels, has not yet been worked out. However, the structural evidence suggests a vesicular packaging and secretory mechanism for antibody release that is consistent with the experimental findings from the studies described in this report.

Hybridomas synthesize and secrete monoclonal antibodies. These cells must express and utilize proteins and physical mechanisms to carry out this process. The precise details of the mechanisms and the identification of specific proteins involved in carrying out this process have not yet been completely elaborated. One aspect of antibody secretion that seems obvious, but for which there is very little direct evidence, is that, in vivo, plasma cells must have an antibody reserve for the eventuality that antigen-specific cells will encounter their particular antigen. It is also possible that the secretory process responds to stimuli other than antigen binding. For example, catecholamines may play a role in stimulating secretion of cytokines, antibodies, or more catecholamines during an inflammatory reaction. To define this process more precisely and to determine whether ENaC plays a role in the process, we measured monoclonal antibody secretion from hybridomas at 10-min intervals before and after treatment with secretory agonists. We also measured whole cell ENaC currents in hybridomas treated with the same agonists.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Plasma cell isolation. Single-cell suspensions were made from the spleens of C57BL6 mice. Spleen cells were depleted of red blood cells by treatment with an ammonium chloride-containing buffer. Recovered lymphoid cells were incubated with a mixture of anti-IgM-FITC (Southern Biotechnology, Birmingham, AL) and anti-syndecan-1-PE (PharMingen, San Diego, CA). The treated lymphoid cells were subsequently selected for IgM+,-Syndecanhigh cells by using a MoFlo high-speed Fluorescent Antibody Cell Sorter (FACS; Cytomation). The recovered cells were placed in serum-containing medium and stored on ice before being used for whole cell patch clamp.

Whole cell patch clamp. Micropipettes were constructed by 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 (in mM) 100 K-gluconate, 30 KCl, 10 NaCl, 20 HEPES, 0.5 EGTA, <10 free Ca2+, and 4 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 (133 mM Na+, 5.3 mM K+, 108.3 mM Cl-). The solutions approximate the ionic gradients across the cell membrane in vivo with a Nernst potential of -83 mV for potassium. Pipettes were mounted in a holder and connected to the head stage 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) by 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 produced a sudden increase in capacitance with no change in seal resistance. The magnitude of the capacitance increase is a direct function of the membrane available to be voltage clamped (i.e., the membrane area and, hence, cell size). Typically, this capacitance was between 5-10 pF for activated peripheral blood lymphocytes. The average capacitances for collecting duct cells were 11 ± 1.4 (rat) and 9 ± 1.7 pF (rabbit).

Previous measurements of transmembrane voltage showed that 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 800 ms between each test voltage. This procedure provided voltages sufficient to measure inward sodium (at more hyperpolarized potentials) and outward potassium currents (at more depolarized potentials). The currents were recorded digitally and filed in real time. The entire procedure was performed by using a DOS Pentium computer modified for analog-to-digital (A/D) signals with pCLAMP 6 software, with an A/D interface controlled by pCLAMP (Axon Instruments, Sunnyvale, CA).

Measurement of monoclonal antibody secretion. The hybridoma cells chosen for these experiments (DB9G8, ATCC no. HB124) secreted monoclonal antibodies directed against insulin. Thus the concentration of these monoclonal antibodies was readily measurable by ELISA, using plates coated with insulin. ELISAs were performed to measure the concentration of monoclonal antibodies from a known quantity of cells into a single volume of medium at 10-min intervals for 60 min. To perform this analysis, we filled 96-well Immunlon plates with an insulin solution (1 mg/ml in PBS) and allowed them to incubate for 24 h. After 24 h, the plates were washed and stored for use over 3 days. For the direct ELISA, each well was filled with 100 µl of blocking buffer (PBS supplemented with 10% FBS). Supernatant samples (200 µl) were obtained at each time point and serially diluted. After an incubation period of 2 h at room temperature, the plates were washed three times with 0.05% Tween 20 in PBS. After washing, the wells were filled with 100 µl of blocking buffer and incubated for 10 min at room temperature. After further washing, alkaline phosphatase-conjugated anti-mouse antibodies (1:1,200 dilution in blocking buffer; Jackson ImmunoResearch Labs) were added to the wells (100 µl). The plates were incubated at room temperature for 30 min. After incubation, the plates were washed three times, and 100 µl of p-nitrophenylphosphate was added to develop the reaction. After 10 min of development, the plates were read for the intensity of the reaction by using a Bio-Rad model 550 microplate reader at a wavelength of 405 nm. On each plate, a standard serial dilution was performed by using an initial concentration of 1 µg/ml of commercially available purified anti-insulin monoclonal antibodies (TaKaRa Biomedicals, Otsu, Shiga, Japan). Concentrations from the supernatant were determined by comparison of reaction intensity with that of the known standard. Though each test sample was serially diluted (1:2), for internal control of the concentration measurements, only the concentration determined from the undiluted sample (for each time point) was used for the analysis of the findings. One technical difficulty was that the antibodies used for the standard curves lost potency daily after reconstitution. This technical difficulty did not affect our ability to detect agonist-induced increases in antibody secretion. However, determinations of the absolute concentration of antibodies from day to day and different experimental protocols performed with the use of different standard antibody lots were subject to considerable variability. For this reason, all experiments were performed with internal controls and no conclusions could be drawn concerning the absolute rate of antibody secretion, precluding comparative analysis of some of the experimental findings.

To measure monoclonal antibody secretion, we placed 5-10 million hybridoma cells in a 15-ml conical centrifuge tube. These were washed in RPMI culture medium supplemented with 25% FBS to remove the antibodies secreted when the cells were in culture. The cells were resuspended in 10 ml of RPMI culture medium supplemented with 25% FBS. Initially, a 50-µl sample of the resuspended cells was removed. The cells were stained (trypan blue) and placed in a hemocytometer, and the viable cells were counted. This procedure was repeated for each time point (0, 10, 20, 30, 40, 50, and 60 min). Also, at each time point, a 200-µl sample was removed and the supernatant was stored for ELISA analysis after completion of the full 60 min. The 200-µl samples were not replaced. This procedure potentially introduced a 2% volume concentration increase for each sample. Between time points the cells were allowed to settle without centrifugation so as not to produce artifactual antibody secretion due to excessive handling. The entire sampling process took <30 s for each time point. An additional 5-10 s was required for the addition of agonists as the 20 min time point.

For assays in which the effect of agonists was to be tested, the agonists [final concentrations: 40 µM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP), 30 µM norepinephrine, or 1.5 µg/ml anti-IgM] were added to the cell suspensions immediately after the 20-min samples were obtained. After each sample, the cells were placed in an incubator at 37°C and allowed to settle. Before each subsequent sample was obtained, the cells were mixed in the suspension by inverting the centrifuge tube three to four times. The entire procedure was then repeated a minimum of six times for unstimulated cells, for each agonist, and for each agonist in the presence of amiloride. For the amiloride trials, RPMI culture medium (25% FBS) was supplemented with the final concentration of amiloride to be tested. The initial cell wash was with amiloride-supplemented RPMI. Thus the cells were continuously exposed to amiloride for the entire procedure, and amiloride was present before the addition of any agonists. All statistical results are presented as means ± SD. Statistical significance was assessed by using Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Plasma cells and hybridomas have more basal ENaC current than nonsecreting lymphocytes. Freshly isolated mouse plasma cells (i.e., antibody-secreting cells) and transformed murine hybridomas (antibody-secreting cells) were whole cell patch clamped with the use of normal ionic gradients to determine whether fusion and transformation induced differences in the amiloride-sensitive inward sodium currents mediated via ENaC. We found that the average ENaC currents observed in the transformed hybridomas (ATCC no. HB124) and the freshly isolated plasma cells were not significantly different (Fig. 1). ENaC inward sodium currents were present in both cell types without the need for agonist stimulation. These findings are in contrast to observations made in whole cell patch-clamped transformed or freshly isolated human and murine B lymphocytes (Fig. 1). The only exception to these observations in nonsecreting B lymphoid cells are lymphocytes derived from individuals with Liddle's disease (3, 5), which have constitutively activated basal ENaC currents due to a premature stop polymorphism of the beta -subunit.


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Fig. 1.   A: typical whole cell clamp records from a hybridoma secreting anti-insulin monoclonal antibodies (MAb) and a transformed human lymphocyte that has no known secretory function (Daudi). B: average current-voltage relations (n = 6 for each cell type) indicate a shift to the right in the reversal potential for the hybridomas compared with the Daudi cells (left). The average inward current (n = 6 for both cell types) is significantly (P < 0.05) increased at a clamp potential of -80 (right).

When the plasma cells and hybridomas were not exposed to any stimuli, the inward amiloride-sensitive sodium current was approximately fivefold larger (P < 0.0001) than in the nonsecreting Daudi lymphocytes (Fig. 1B, right). Figure 1A depicts representative whole cell currents from a hybridoma, a Daudi lymphocyte, and a freshly isolated plasma cell. The most prominent difference between these records is the presence of inward currents in the plasma cell and hybridoma records and the absence of these currents in the Daudi lymphocyte record. The presence of these currents caused the plasma cells and hybridomas to be depolarized by 30-50 mV, as evidenced by the average current-voltage relations shown in Fig. 1B, left. This depolarization is caused by a relative increase in sodium permeability (PNa/PK) in the hybridomas. Whereas plasma cells also secrete antibodies, it is unlikely that the rate of production and secretion is comparable to that of hybridomas; thus the relative sodium permeability increase over Daudi cells is not as pronounced and the reversal potential is less depolarized. A significant difference between the plasma cells and hybridomas and the Daudi lymphocytes is that the plasma cells and hybridomas secrete antibodies and the Daudi cells do not. Thus the fivefold difference in sodium conductance may be related to the physiological mechanisms involved in antibody secretion. This potential link between enhanced plasma membrane sodium permeability and the secretory process is further supported by the specificity of the conductance increase. The 50-mV shift in the reversal potential indicates that the difference between the hybridomas and the Daudi cells is not a general increase in membrane permeability but, rather, a specific increase in ENaC conductance. It is also unlikely that the difference is related to species (mouse vs. human), because we have demonstrated previously that mouse lymphocytes and human lymphocytes have similar basal whole cell currents (4) that are indistinguishable from those shown in the Daudi lymphocyte record. To further our understanding of this phenomenon, we performed whole cell patch clamp on hybridomas to test the hypothesis that secretory agonists activated ENaC.

Plasma cell and hybridoma ENaC current is increased by agents that also provoke antibody secretion. Antigen binding produces a signal recognized by B lymphocytes and plasma cells by cross-linking surface IgM. We hypothesized that if ENaC played a role in antibody secretion, cross-linking surface IgM with anti-IgM antibodies should increase the amount of ENaC current in whole cell clamped hybridomas and plasma cells. Figure 2 shows the agonist effect of anti-IgM antibodies on the inward sodium current in a plasma cell and a hybridoma. The qualitative and quantitative effects of this treatment were not different between the cell types. Thus hybridomas appear to be an appropriate model cell type for investigation of the role of ENaC in the process of regulated antibody secretion because there are no differences between freshly isolated plasma cells and hybridomas with respect to either basal or agonist-stimulated ENaC currents.


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Fig. 2.   Whole cell patch-clamp current records show that in the freshly isolated untransformed plasma cells, cross-linking surface IgM with anti-IgM antibodies specifically increases the inward sodium current (amplitude). The agonist effect on ENaC currents appeared in <1 min after antibodies were added to the bath solution. Normal ionic gradients for sodium, potassium, chloride, and calcium were used to obtain these whole cell clamp current records.

We have shown previously that cAMP activates ENaC in lymphocytes (2-6) and that norepinephrine activates lymphocyte ENaC via the alpha 1-adrenergic receptor and increases in cellular cAMP (2). Therefore, we used these ENaC agonists to determine whether the same amiloride-sensitive current was activated in hybridomas. These experiments were necessary because it is possible that all of the ENaC expressed was activated in these cells, based on the significantly increased basal amiloride-sensitive current (Figs. 1 and 2). Figure 3 shows that treatment with either a membrane-permeant cAMP analog (8-CPT-cAMP) or norepinephrine increased the inward current and that all of the agonist-activated current was inhibited by amiloride.


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Fig. 3.   Whole cell clamp records show that agonists 8-CPT-cAMP (A), norepinephrine (B), and anti-IgM (C) produce the same increase in inward ENaC current in hybridoma cells secreting MAbs to insulin. In each case the agonist-activated inward current is completely and specifically inhibited by amiloride.

Note that the physical forces involved in sealing patch pipettes to cells, and the subsequent formation of the whole cell configuration, can induce variability in the transmembrane currents and, therefore, introduce some variability to these measurements. However, care was taken with every whole cell preparation to avoid any obviously inconsistent currents.

Because antigen binding can induce antibody secretion in situ, we hypothesized that cross-linking surface IgM (to mimic antigen binding) might increase amiloride-sensitive current if ENaC were involved in the secretory process. To test this hypothesis, we whole cell clamped hybridomas as described. After basal currents were measured, the cells were superfused with antibodies directed against surface IgM (1.5 µg/ml). Figure 3C shows that this treatment produced the same increased current response as treatment with norepinephrine or 8-CPT-cAMP. Thus these electrophysiological findings are consistent with the hypothesis that ENaC plays a role in antigen-provoked antibody secretion by hybridomas. Figure 4 shows the average significant increase in inward sodium current at the equilibrium potential for potassium (to eliminate any potential current carried by potassium) for each of the ENaC agonists.


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Fig. 4.   Average inward current at the equilibrium potential for potassium (-80 mV) is significantly increased by agonists 8-CPT-cAMP (A), norepinephrine (Norepi, B), and anti-IgM (C). *P < 0.05.

Antibody secretion is acutely increased by agents that activate ENaC current in hybridomas. Whole cell clamp analysis of hybridoma ENaC current showed that various agents acutely increased ENaC. We hypothesized that these same agents could provoke a secretory response by these hybridomas. To test this hypothesis, we used direct ELISA analysis to measure the change in concentration of secreted monoclonal antibodies at 10-min intervals. Between 3 and 5 million cells per assay were washed in fresh culture medium and resuspended in 10 ml of antibody-free culture medium in 15-ml conical centrifuge tubes. The cells were allowed to settle without centrifugation and were kept at 37°C in a cell culture incubator. After 10 min, a sample of the supernatant was taken for ELISA analysis, the cells were resuspended, and a second sample was taken for cell counts and viability. This process was repeated every 10 min for 60 min. By following this procedure, a basal secretion rate was established, as shown in Fig. 5A. Figure 5B shows that there was no change in cell number and viability. Also, there was no change in the cell number or viability of the cells in any of the experiments in which the cells were treated with agonists (not shown).


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Fig. 5.   A: change in supernatant anti-insulin MAb concentration with time. The slope of the line is the basal rate of MAb secretion. The average rate of secretion is 5.8 ± 3.4 ng · ml-1 · 106 cells-1 · min-1. This value was calculated from the linear regression slopes of 10 repetitions of the control experiment. The level at the 10 min time point is high relative to the rate due to antibody secretion in response to the physical stimulation of washing the cells. B: viable cell counts taken at each time point using trypan blue. Identical cell count results were obtained when agonists were added after the 20 min sample to provoke secretion.

Once the basal secretion rate was established, we attempted to alter the rate by adding the agonists that increased plasma membrane ENaC current. To test the hypothesis that these potential "secretagogues" acutely increased MAb secretion, we used the following procedure. Immediately after the 20-min samples were obtained, we added 8-CPT-cAMP (40 µM), norepinephrine (30 µM), or anti-IgM (1.5 µg/ml) to the cell suspensions. Subsequent to agonist addition, samples were taken in the same manner as described above. We found that the addition of 8-CPT-cAMP or anti-IgM provoked a significant increase in the MAb concentration at the 30 min time point, and there was no further increase in the concentration at any subsequent time point. The same pattern was observed with norepinephrine; however, the significant increase was observed at the 40 min time point. These findings are shown in Fig. 6.


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Fig. 6.   With each agonist tested [8-CPT-cAMP (A), norepinephrine (Norepi, B), and anti-IgM (C)], addition of the agonist provoked a significant increase in the supernatant MAb concentration in the time points following addition of the agonist. In the case of norepinephrine (B), the effect was delayed until the second time point after the addition. After the initial large secretory response, there was no further increase in the MAb concentration. nsd, No significant difference.

Amiloride inhibits agonist-induced antibody secretion. As an additional direct test of the hypothesis that ENaC sodium channels play a role in agonist-provoked secretion of MAbs, we determined whether agonist-provoked secretion could be inhibited with the ENaC-specific inhibitor amiloride. The experimental procedure was the same as that described earlier. However, the cells were washed in medium supplemented with amiloride (2, 20, and 100 µM). The amiloride remained present for the entire sampling procedure. These findings are shown in Fig. 7. Interestingly, 2 µM amiloride did not prevent the agonist-provoked increase in secretion but, rather, delayed the effect by one time point to 40 min instead of 30 min (Fig. 7A). This finding is significant because the IC50 for amiloride is 75 nM, and a concentration of 2 µM is more than sufficient to inhibit all of the ENaC current (see Fig. 2). Thus it does not appear as though the plasma membrane ENaC conductance is necessary for regulated secretion to occur. However, a concentration of 20 µM amiloride inhibited all of the agonist-provoked increase in MAb secretion (Fig. 7B), and 100 µM amiloride inhibited all MAb secretion (Fig. 7C). Because amiloride specifically inhibits ENaC at concentrations of 20 µM and below, it is likely that the delay of secretion at 2 µM and the complete inhibition of the agonist-provoked secretion at 20 µM are due to inhibition of ENaC. However, at a concentration of 100 µM, amiloride may have less specificity. Thus it is not possible to conclude that at this high concentration, inhibition of ENaC exclusively by amiloride blocks all MAb secretion by hybridomas. These findings indicate that the plasma membrane ENaC-mediated plasma membrane Na+ conductance played no role in regulated secretion. However, because amiloride did inhibit regulated secretion at relatively high concentrations, it is possible that inhibition of ENaC in the membranes of secretory vesicles within the cytoplasm was the mechanism of inhibition.


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Fig. 7.   Inhibitory effect of amiloride on agonist-induced MAb secretion. The inhibition of cAMP-induced secretion is shown for 3 amiloride concentrations: 2 µM (A), 20 µM (B), and 100 µM (C). Identical results were obtained when norepinephrine or anti-IgM was used to stimulate secretion (not shown).

Uncharged amiloride inhibits agonist-provoked MAb secretion. Because the inhibition of secretion by amiloride followed the ENaC inhibition curve with respect to the uncharged amiloride concentration, the findings suggested that the inhibition of regulated secretion by amiloride was due to inhibition of ENaC within the cell. To test this hypothesis further, we altered the uncharged amiloride concentration by varying the extracellular pH. The pKa of amiloride is 8.7. Thus amiloride acts as a weak base, and the uncharged amiloride concentration is determined by the Henderson-Hasselbalch equation. For any concentration of amiloride, the uncharged component will be reduced as the pH is reduced and increased at higher pH values. For these experiments we used culture medium pH values of 6.6, 7.4, and 8.2. The total amiloride concentration was fixed at 2 µM. Thus the uncharged (membrane permeant) amiloride concentrations were 16, 100, and 632 nM at the respective pH values. These concentrations span the amiloride-binding curve, which has an IC50 of ~75 nM. To ensure that the pH had no independent effect on agonist-stimulated secretion, we performed control experiments without amiloride supplementation. Figure 8 shows the findings when the pH was elevated to 8.2. There was a qualitative difference in the findings compared with experiments performed at a pH of 7.4. The lower absolute concentration of antibodies measured at pH 8.2 may have resulted from differences in standard antibody efficacy or from differences in the cells secretion rate. The lot-to-lot differences in efficacy of the anti-insulin MAbs used for the standard curves performed with each experiment, as well as their tendency to lose potency within a short time (2 wk), preclude comparisons of absolute antibody concentration. Thus all experiments were performed using internal controls. This technical difficulty does not, however, alter the ability to detect the effects of agonists to activate antibody secretion and does not alter the conclusions based on these findings.


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Fig. 8.   Secretion assays performed as a control for the effect of extracellular pH show that an increase in the extracellular pH to 8.2 had no effect on the ability of 8-CPT-cAMP to provoke a rapid secretion of MAbs.

Figure 9 shows the results obtained when amiloride (2 µM total concentration) was added to the culture medium. At pH 6.6, 2 µM amiloride (16 nM uncharged) had no effect on agonist-induced secretion (Fig. 9A). At pH 7.4 [2 µM amiloride (100 nM uncharged), close to the IC50], there was partial inhibition (not shown). When the pH was increased to 8.2 [2 µM amiloride (632 nM uncharged)], agonist-induced MAb secretion was completely inhibited (Fig. 9B). These findings precisely follow the amiloride-binding curve for ENaC, using the uncharged component of amiloride.


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Fig. 9.   Effect of varying the external pH on the ability of amiloride to inhibit regulated secretion stimulated by 8-CPT-cAMP. The pKa of amiloride is 8.7. Therefore, at an external pH of 6.6, the uncharged (membrane permeant) concentration of amiloride was 16 nM. Under these conditions, amiloride failed completely to inhibit regulated secretion (A). When the external pH was raised to 8.2 (uncharged amiloride concentration = 632 nM), amiloride inhibited regulated secretion completely (B). The uncharged amiloride concentration precisely follows the amiloride-binding curve for amiloride inhibition of ENaC. A partial inhibition by 2 mM amiloride was observed when the pH was 7.2. This partial inhibition was manifested as a delay in the response to cAMP and can be seen in Fig. 7A.

Because amiloride acts as a weak base, it is possible that amiloride could have induced a change in the cytosolic pH, which indirectly affected agonist-induced MAb secretion. To test this possibility, we repeated the experiment using ammonium, which is also a weak base with a pKa similar to that of amiloride. Thus a culture medium pH of 8.2 and an ammonium concentration of 3.2 µM produced an uncharged ammonia concentration of 632 nM, the same as the uncharged amiloride concentration that completely inhibited agonist-induced MAb secretion under identical conditions. Figure 10 shows the findings from these experiments. Ammonium had no effect on the secretory response of the hybridomas. Therefore, we could rule out the hypothesis that indirect pH changes inhibited the secretory process. Taken together, these findings consistently support the notion that amiloride inhibition of ENaC within the cell is responsible for the inhibition of agonist-stimulated MAb secretion. These findings also indicate that the Na+ channel function of ENaC within the plasma membrane does not play a role in this process, because 2 µM total amiloride concentration at any pH was sufficient to inhibit all of the Na+ current. Clearly, this concentration had no effect on secretion at pH 6.6. 


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Fig. 10.   To test the hypothesis that because amiloride is a weak base and, therefore, was changing the cytosolic pH to produce the inhibition of secretion, we substituted ammonium (at an equivalent uncharged concentration) for amiloride. Results show that ammonium had no effect, thereby ruling out the possibility that amiloride-induced pH changes inhibited regulated secretion.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Whole cell patch clamp of plasma cells and hybridomas cells has shown that basal inward sodium current is present in the absence of any stimulation with ENaC agonists. Also, we have shown that cross-linking surface IgM with anti-IgM antibodies increases the ENaC-mediated inward sodium current specifically. Both cell types synthesize and secrete antibodies. These findings are in contrast to whole cell patch-clamp analysis of B lymphoid cells that were not actively synthesizing and secreting antibodies (1-5). This difference led to the hypothesis that ENaC may play a role in the regulated secretion of antibodies. Because regulated secretion must involve the movement of antibody containing vesicles into the plasma membrane, one explanation for the observation of increased ENaC current is that the secretory vesicles sequestering antibodies and containing ENaC in the membrane portion cycle into and out of the plasma membrane to accomplish antibody release. Thus at the instant the cells were whole cell clamped, there was active ENaC in the plasma membrane that would not be present if this process were not taking place. Because no differences in ENaC current (either basal or in response to agonist-mediated activation) were observed between untransformed plasma cells and hybridomas, it appears as though the fusion and transformation used to produce hybridomas did not directly alter the cell physiological process to be examined. Because it is not possible to use plasma cells to perform the secretion measurements necessary to test this hypothesis (because one cannot obtain enough fresh plasma cells that secrete antibodies to a known substrate), hybridomas that secrete MAbs to insulin were chosen to test the hypothesis. Note that the difference in basal ENaC current between antibody-secreting and nonsecreting B lymphoid cells suggested the hypothesis to be tested, but a comparison of ENaC currents between secreting and nonsecreting B lymphoid cells could not in itself provide sufficient information to test the hypothesis.

The secretion assay and the correlated electrophysiological experiments presented here directly tested the hypothesis that ENaC plays a role in a specialized lymphocyte secretory function, i.e., secretion in response to agonist stimulation. This hypothesis was formed to address the question of the utility of ENaC in lymphocyte function. Previously we have demonstrated expression of ENaC by normal and polymorphic lymphocytes (5). We have shown that lymphocyte ENaC is hyperactive in Liddle's disease (5, 6, 9). We have also demonstrated activation of lymphocyte ENaC by norepinephrine, via the alpha 1-adrenergic receptor (2). We know that various signal transduction pathways alter the activity of ENaC in lymphocytes. For example, ENaC is activated by cAMP (2, 3, 5), modification of G proteins by pertussis toxin activates lymphocyte ENaC (4), and aldosterone acutely activates human lymphocyte ENaC via a transmethylation reaction (13). However, none of these previous findings provides an answer to the question, "What is the physiological function of ENaC expressed by lymphocytes?"

The physiological function of ENaC is understood in the context of the regulation of salt balance. ENaC is expressed by renal principal cells in the distal nephron. The role of ENaC in the kidney is well understood. The most direct evidence for its role as a regulator of sodium reabsorption is the excessive salt and water accumulation associated with Liddle's disease, where there is a proven gain of function polymorphism in the beta -subunit of ENaC (12). Another clear indication of its renal function is the effect of amiloride. This compound specifically inhibits ENaC and clinically is used as a potassium-sparing diuretic. Also, amiloride completely ameliorates hypertension in individuals with hyperactive ENaC (6).

In lymphocytes, ENaC clearly functions as a sodium channel. However, it is unlikely that lymphocyte ENaC plays a role in salt and water regulation, as it does in renal principal cells. To our knowledge, lymphocytes do not influence the regulation of salt balance in any way. Thus two possibilities are that either the role of ENaC in lymphocyte cell physiology remains to be defined or ENaC plays no role at all in lymphocyte cell physiology. To distinguished between these possibilities, we examined the most obvious (to us) function of lymphocytes. These cells synthesize proteins, such as antibodies and cytokines, for use in mediating inflammatory reactions. We surmised that during an inflammatory reaction to an acute infection, lymphocytes would have to secrete their products very rapidly in a circumscribed area to produce the most efficient reaction in the shortest time. This made sense because bacteria, for example, can multiply at a rapid rate. Thus, to overcome a bacterial infection, the rate of secretion of inflammatory mediators must exceed the rate at which the bacteria multiply. Also, it seemed logical to assume that secretion of these mediators should increase specifically by interaction with antigen or other inflammatory mediators. If this were not the case, the circulation would be inundated with antibodies and cytokines. Using these assumptions and observations, we chose to test directly whether we could alter the rate of monoclonal antibody secretion by hybridoma cells. These cells provide the advantage of secreting large quantities of a single known antibody. This property made it possible to reproducibly measure the rate of secretion at 10-min time intervals by direct ELISA. Figures 4-6 show that the inherent variability of ELISA analysis can be overcome to a degree sufficient to demonstrate significant increases in MAb secretion in response to various chemical stimuli.

Figure 2 shows three stimuli that increase ENaC current in the plasma membrane. These same stimuli acutely increased the rate of antibody secretion (Fig. 5). In fact, these stimuli induced the cells to secrete a pool of preformed antibodies, possibly destined for acute secretion within 10 min, because the secretion rate fell to zero after the single large increase in the rate immediately subsequent to the addition of the agonists. This correlation between currents and the secretory response is suggestive but does not directly implicate ENaC in the process. However, the findings shown in Figs. 6 and 8 directly implicate ENaC in the process, because amiloride inhibits the process.

The ability of amiloride to inhibit regulated secretion partially at 2 µM and completely at 20 µM suggests that the inhibition was intracellular, rather than an inhibition of current at the plasma membrane. The IC50 for amiloride interaction with ENaC is ~75 nM. We have routinely used 2 µM amiloride to completely inhibit ENaC current in lymphocytes in a number of previous studies (1-6). Figure 2 shows the complete inhibition of ENaC current in hybridomas by 2 µM amiloride. Clearly, 2 µM amiloride does not completely inhibit agonist-stimulated secretion (Fig. 6). However, it does delay the effect, as could be expected from a submaximal concentration. The pKa of amiloride is 8.7. Thus, from the Henderson-Hasselbalch equation, the uncharged form of amiloride at pH 7.4 (the pH of the culture medium) is 100 nM. This concentration of amiloride produces a 60% inhibition of ENaC single-channel current in outside-out patches (13). Following the same reasoning, when the medium contained 20 µM amiloride, the uncharged amiloride concentration was 1 µM. This concentration is sufficient to inhibit all ENaC current. Figure 7 shows that 20 µM amiloride prevented the agonist-stimulated MAb secretion.

By varying the pH of the external solution, we were able to use a single concentration of amiloride (2 µM) and alter the inhibition of regulated secretion. Control experiments ruled out the possibility of direct effects of varying the external pH on agonist-stimulated increases in antibody secretion. Additional control experiments using ammonium ions at an equivalent unionized concentration demonstrated that a potential cytoplasmic pH change had no effect on regulated secretion. The remaining possibility was that amiloride inhibited ENaC within the cell. Thus vesicles containing MAbs must have ENaC in their membrane, and this "vesicular" ENaC must play a role in the process of acute antibody secretion that is stimulated by catecholamine or antigen binding. This function may be unique to lymphocytes, or it may also be present in other types of cells that secrete various products in response to external stimuli.

The inhibition of regulated secretion may be due to inhibition of ENaC sodium current across the vesicular membrane or may be due simply to steric interference in a possible binding function of ENaC. Unfortunately, at the present time we are not able to distinguish between these two possible inhibitory mechanisms. It is possible that the Na+ channel function of ENaC is essential for some aspect of the secretory process and that blocking this function on secretory vesicles within the cytoplasm inhibits secretion. It is also possible that ENaC serves as a binding partner for other proteins involved in the movement, fusion, or opening of the secretory vesicles and that when amiloride is bound to ENaC, there is a steric inhibition that prevents a required physical interaction. At present we are not aware of an experiment that would distinguish between these possibilities. Nevertheless, the present observations are consistent with, and support, the hypothesis that ENaC is involved in the specialized process of regulated antibody secretion.

Finally, these observations suggest an immunologic role for ENaC in inflammatory reactions because amiloride can inhibit antibody secretion in vitro. Amiloride is currently used clinically as a potassium-sparing diuretic. Thus the question arises as to whether individuals on amiloride therapy are immunologically compromised. We think this possibility is unlikely because the concentrations of amiloride necessary to inhibit regulated secretion at a physiological pH are well in excess of the therapeutic range of amiloride. At normal serum pH it would take an amiloride concentration in excess of 10 µM to inhibit secretion. This concentration is well above the concentration of amiloride necessary for clinical effectiveness as a potassium-sparing diuretic. Thus it is unlikely that amiloride therapy would have any effect on immune cell function via blockade of ENaC.


    ACKNOWLEDGEMENTS

We thank Drs. John F. Kearney and Kalaya Attanavanich for the gift of FACS-sorted murine plasma cells.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-52789 (J. K. Bubien). J. K. Bubien is an Established Investigator of the American Heart Association.

Address for reprint requests and other correspondence: J. K. Bubien, Dept. of Physiology and Biophysics, 726 McCallum Bldg., Univ. of Alabama at Birmingham, Birmingham, Alabama 35294-0005 (E-mail: bubien{at}uab.edu).

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. Section 1734 solely to indicate this fact.

July 24, 2002;10.1152/ajpcell.00175.2002

Received 16 April 2002; accepted in final form 17 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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