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Am J Physiol Regul Integr Comp Physiol 290: R1183-R1190, 2006. First published November 17, 2005; doi:10.1152/ajpregu.00716.2005
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Neurohypophyseal Hormones: From Genomics and Physiology to Disease

IL-1beta directly excites isolated rat supraoptic neurons via upregulation of the osmosensory cation current

Yassar Chakfe ,* Zizhen Zhang,* and Charles W. Bourque

Centre for Research in Neuroscience, Montreal General Hospital and McGill University, Montreal, Quebec, Canada

Submitted 7 October 2005 ; accepted in final form 9 November 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have shown that IL-1beta can excite the magnocellular neurosecretory cells (MNCs) of the hypothalamus. However, it is not known whether IL-1beta can have direct IL-1 receptor type 1 (IL-1R1)-mediated effects on MNCs, and little is known about the cellular mechanisms by which IL-1beta influences electrical activity in these cells. Here, we used patch-clamp recordings to examine the effects of IL-1beta on acutely isolated rat MNCs. We found that IL-1beta directly excites MNCs in a dose-dependent manner and that this response can be blocked by an inhibitor of the IL-1R1. Voltage-clamp analysis of the current evoked by IL-1beta revealed a linear current-voltage relationship between –90 and –20 mV, and a reversal potential near –35 mV. This value was not affected by reducing the concentration of chloride ions in the external solution, indicating the involvement of a nonselective cation conductance. The effects of IL-1beta were inhibited by Na-salicylate, an inhibitor of cyclooxygenase. Moreover. the effects of IL-1beta were mimicked and occluded by PGE2, and were inhibited by AH-23848, an antagonist of the PGE2 type 4 (i.e., EP4) receptor. The current evoked by IL-1beta was also abolished by 100 µM gadolinium (Gd3+), but was significantly larger when examined in cells preshrunk by negative pressure applied via the recording pipette. IL-1beta alone did not cause changes in cell volume nor in the mechanosensitivity of MNCs. We conclude that IL-1beta directly excites MNCs via an IL-1R1-mediated induction of PGE2 synthesis and EP4 receptor-dependent autocrine upregulation of the nonselective cation conductance that underlies osmoreception.

vasopressin; prostaglandin; EP4 receptor; osmosensitivity; neurohypophysis

RELEASE OF THE INFLAMMATORY cytokine IL-1beta into the circulation is believed to recruit central neuronal responses that mediate host defense through a cascade of events that is initially triggered in brain vascular cells. Specifically, IL-1beta binding to the IL-1 receptor type 1 (IL-1R1) expressed in vascular cells is believed to induce the expression of cyclooxygenase-2 (COX-2) activity (15), thereby increasing the synthesis and release of PGs. PGs can then diffuse across the blood-brain barrier to induce neuronal responses (e.g., 25). However, previous studies have shown that IL-1beta is also released within the brain parenchyma during systemic infection (e.g., 23, 33) and in response to traumatic injury (e.g., 19) or ischemia (e.g., 6). During systemic infection, high levels of IL-1beta are observed predominantly in the hypothalamus where, together with vascular IL-1beta, they may initiate host defense responses through alterations in behavioral and neuroendocrine function. For example, hypothalamic IL-1beta has been implicated in the mediation of sickness behavior (2), loss of appetite (17), fever (9), and activation of the hypothalamo-pituitary-adrenal axis (32) and in the release of oxytocin (29) and vasopressin (16, 31, 36) from the neurohypophysis. The pressor and antidiuretic effects associated with vasopressin release are particularly important during the early phase of septic shock, because they may help maintain tissue perfusion under conditions where powerful vasodilatory substances are being released (13).

Recent studies have provided important insight into the cellular mechanisms by which IL-1beta may increase vasopressin secretion from the neurohypophysis. Indeed, recordings from hypothalamic slices have shown that IL-1beta can depolarize magnocellular neurosecretory cells (MNCs) in the supraoptic (18) and paraventricular nuclei (10). The depolarizing effects of IL-1beta observed in these studies were insensitive to tetrodotoxin and were blocked by inhibitors of COX, suggesting that they were mediated postsynaptically, through a mechanism involving the production of PGs. Moreover, whole cell patch-clamp recordings have shown that both IL-1beta (10) and PGs (10, 28, 30) depolarize MNCs through the activation of a nonselective cation conductance. Taken together, these findings support the hypothesis that IL-1beta may induce vasopressin release by exciting MNCs through a mechanism that involves the production of PGs (18). However, the identity of the cells responsible for detecting IL-1beta and producing PGs remains unknown. Indeed, because PGs diffuse freely across cell membranes (9, 26), their release is regulated primarily by the rate of synthesis rather than by action potential-dependent exocytosis. Thus the effects of bath-applied IL-1beta observed on MNCs in the presence of tetrodotoxin in brain slices could have involved PGs produced either by adjacent cells (i.e., via a paracrine effect), or by the MNCs themselves (i.e., via an autocrine effect). Although two immunocytochemical studies have shown that the IL-1R1 receptor responsible for mediating the effects of IL-1beta is expressed in hypothalamic nuclei containing MNCs (8, 15), these studies have provided contradictory results concerning the presence of such receptors on the somata of MNCs. In this study. therefore. we examined the effects of IL-1beta on acutely isolated MNCs, under experimental conditions that eliminate the involvement of potential paracrine effects.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of MNC somata. MNC somata were isolated from brains of male Long-Evans rats (100–200 g) killed by decapitation as previously described (5, 35) and in accordance with a protocol approved by the Animal Care Committee of McGill University. Briefly, coronal slices of hypothalamus (about 1-mm thick) were obtained and blocks (~1 mm3) containing the supraoptic nucleus were removed with the use of iridectomy scissors. Tissue blocks were incubated for 90 min at 33°C in 10 ml of an oxygenated (100% O2; pH 6.9–7.0) PIPES saline comprising (in mM): 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 20 PIPES (disodium salt), 25 D-glucose, as well as 0.7 mg/ml trypsin (type XI; Sigma, St. Louis, MO). Blocks were subsequently washed in trypsin-free oxygenated PIPES saline (pH 7.3–7.4) and kept (<8 h) until use. When required, blocks were triturated with fire-polished Pasteur pipettes (0.2–0.5 mm ID), and the suspension was plated onto 35-mm petri dishes (Corning Costar, Cambridge, MA).

Whole cell recording. Dishes were mounted onto the stage of an inverted-phase contrast microscope and perfused (2 ml/min) with a HEPES saline solution (22–24°C; pH 7.35) comprising (in mM): 120 NaCl, 4 NaOH, 3 KCl, 1 MgCl2, 10 HEPES, 1 CaCl2, 10 glucose. Osmolality was adjusted to 295 ± 3 mosmol/kg by the addition of mannitol as necessary. For chloride substitution experiments, this control medium was modified by the replacement of 120 mM NaCl with 60 mM Na2SO4 (low Cl– medium). During voltage-clamp experiments, all media contained 0.5 µM tetrodotoxin. Whole cell recordings were made using pipettes filled with a solution (pH 7.15) comprising (in mM): 150 K-gluconate, 10 HEPES, 1 MgCl2, and 1.6 EGTA. Membrane voltage (dc, 5 kHz) and current (dc, 200 Hz) were recorded through an Axopatch 1D amplifier (Axon Instruments, Union City, CA) and captured using a Digidata 1200B interface driven by Clampex 8 software (Axon Instruments).

Production and measurement of changes in cell volume. In some experiments, we stimulated the stretch-inhibited cation (SIC) channels in MNCs by reducing cell volume via the application of negative pressure (–100 mmHg) via the recording pipette. The amplitude of the suction stimulus was controlled by an air-filled syringe connected to the pipette via Tygon tubing and was monitored through a digital pressure transducer (model PM015D; World Precision Instruments, Sarasota, FL) connected to the tubing via a T-junction. Volume changes (%{Delta}V) were monitored by digital imaging and morphometry, as performed previously (39). Briefly, digital phase-contrast images were captured every 5 s during the experiment, and the perimeter of the cell in each image was traced offline using Scion Image for Windows version 4.02 (Scion, Frederick, MD). The maximal cross-sectional area (CSA; in pixels) was determined by the software. All values of CSA measured in the control period were averaged (CSAo) and values of relative (normalized) volume at each individual time point (nVt) were calculated from the CSA value at that time point (CSAt) using the equation; nVt = [(CSAt)1.5/(CSAo)1.5]. For comparison purposes, changes in cell volume observed at specific time points (e.g., t = 60 s) were converted to %change using the equation %{Delta}V = 100 x (1 – nVt).

Measurement of the mechanosensitivity index. An index of mechanosensitivity (MI) was calculated by dividing the increase in membrane conductance ({Delta}G) provoked per unit change in cell volume. Unit changes in volume observed at time t were measured as 1 – nVt, as indicated above. Changes in conductance observed at time t ({Delta}Gt) were calculated from current responses ({Delta}I) to voltage steps (–40 mV; 500 ms) as {Delta}G = {[{Delta}I/(–0.04)] – Go}, where Go is the average absolute membrane conductance observed during the control period. The MI observed at various times following the onset of the stimulus (MIt) was thus computed as MIt = {Delta}Gt/(1 – nVt ). Values of MIt could be reliably computed for time intervals ranging between 20 and 60 s following the onset of the stimulus. No systematic changes in MIt were observed as a function of time. Moreover values of {Delta}Gt plotted as a function of (1 – nVt) could be fitted by linear regression, confirming the lack of changes in MIt as a function of volume. Thus for each cell, the single value of MI was determined from the average of the values of MIt.

Drugs. Human IL-1beta fragment 163–171 (IL-1beta; Sigma) was dissolved in distilled water at a concentration of 10–4 M and kept frozen until required. The 153-amino acid 17-kDa recombinant human IL-1 receptor antagonist protein (IL-1ra; R&D Systems, Minneapolis, MN) was dissolved at 20 µg/ml in PBS containing 0.2% BSA, and aliquots were stored at –20°C until used. Aliquots of IL-1beta and IL-1ra were thawed and dissolved in HEPES saline solution at the concentrations required before use. Sodium salicylate (Sigma) was directly dissolved into HEPES saline at the concentration required. PGE2 and the EP4 PG receptor antagonist AH-23848 (both from Sigma) were dissolved in DMSO and diluted into perfusion solutions so that the final concentration of DMSO was <0.1%. The effects of IL-1beta in DMSO-containing solutions were not significantly different than those observed in DMSO-free HEPES-based solutions (P > 0.05; data not shown). Drugs were applied either by bath application via perfusion of the whole dish (2–4 ml/min) or by direct perfusion of a single cell with a three-barreled fast solution changer (SF-77B; Warner Instruments, Hamden, CT). In the latter case, the delivery tube (1 ml/min) was placed directly upstream of the cell being recorded (<300 µm away) and no other cell was present upstream of the cell being tested, thereby excluding the possibility of paracrine effects. During direct cell perfusion experiments, complete exchange of the solution perfusing the cell was achieved in less than 20 ms.

Statistics. Values are reported as means ± SE. Comparisons between groups were made using the Student's t-test, the paired t-test, or a Mann-Whitney rank sum test, as appropriate. One-way ANOVA (Sigmastat 2.1; Jandel, San Rafael, CA) was performed for the comparison of multiple groups. Where differences were found, Student-Newman-Keuls test for multiple comparisons was performed post hoc to identify specific distinctions. Differences between means were considered significant when P < 0.05. Fitting of the three-parameter logistic equation through dose-response data points was performed using Sigmaplot 8 (SPSS Science, Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Actions of IL-1beta on isolated MNCs. To define any direct effect of IL-1beta on MNCs, we performed whole cell voltage-clamp recordings from neurons acutely isolated from the supraoptic nucleus of adult rats. Moreover, to avoid the possibility that substances released from other types of cells present in the dish might induce indirect paracrine effects, the fluid perfusion device was positioned so that no other cell was located upstream of that being recorded (see EXPERIMENTAL PROCEDURES). As illustrated in Fig. 1A, sudden exposure (delay <20 ms) to a sustained concentration of IL-1beta induced the appearance of a slowly developing inward current. During these experiments, membrane conductance was measured from the amplitude of current responses to 500-ms hyperpolarizing steps (–40 mV) applied every 5 s. As shown in Fig. 1A, the inward current induced by IL-1beta was accompanied by a progressive and reversible increase in membrane conductance. For each cell tested, we quantified the increase in conductance induced by a specific concentration of IL-1beta by subtracting the baseline conductance of the cell from that observed 120 s following the onset of the application. As plotted in Fig. 1B, different concentrations of IL-1beta induced a dose-dependent increase in membrane conductance, with a half-maximal effect (EC50) at 15 ± 13 nM and an apparent maximum change of +1.3 nS (r2 = 0.80). All cells tested with a concentration of IL-1beta >20 pM (n = 40) showed positive responses to the drug.


Figure 1
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  		Fig. 1. IL-1beta directly activates an increase in membrane current in magnocellular neurosecretory cells (MNCs). A: top trace shows whole cell membrane current in a voltage-clamped isolated MNC (Vhold = –66 mV) exposed to 20 nM IL-1beta (bar) under conditions preventing paracrine effects. Downward deflections are responses to 500-ms hyperpolarizing steps to –106 mV. Bottom: plot of membrane conductance as a function of time for the experiment shown above. Note that IL-1beta induces a progressive and reversible increase in holding current and membrane conductance. The slanted bars indicate a 3-min gap in the recording during which current-voltage relations were measured (not shown). B: plot shows the means ± SE change in conductance observed in different cells as a function of the concentration of IL-1beta ([IL-1beta]). Numbers in parentheses indicate how many cells were tested at each concentration. Solid line is the best fit through the data points according to the logistic equation.

 
We next examined the effects of IL-1beta on isolated MNCs tested under current clamp conditions. Because no differences were noted in the time course and amplitude of responses evoked by direct cell perfusion or bath application of IL-1beta under voltage-clamp (data not shown), the latter (simpler) approach was used in these experiments. As illustrated in Fig. 2A, depolarizing and excitatory responses evoked by concentrations of 40–200 pM reached a peak within 30–60 s and recovered within 0.5–5 min following washout. The excitatory effects of IL-1beta repeated 2–3 times in the same cell following complete washout. Higher concentrations (≥40 nM) produced depolarizing effects that generally did not reverse for the remainder of most recordings (10–30 min). In contrast, applications of IL-1beta at concentrations of 0.04–4 pM were ineffective (n = 6; Fig. 2A), in agreement with the lack of significant effects of the drug on membrane conductance under voltage clamp (Fig. 1B). Finally, to confirm the specificity of these effects, we examined the actions of IL-1beta in the presence of recombinant human IL-1R1 antagonist (IL-1ra). In these experiments, IL-1ra was applied 40–60 s before IL-1beta, and the drug remained present while the agonist was being applied. When present at a concentration of 160 pM, IL-1ra blocked the responses in only two of six cells tested with IL-1beta (400 pM; data not shown). In contrast, when applied at a concentration of 1.2 nM, IL-1ra blocked the excitatory actions of IL-1beta in 7 of 10 cells tested. In the three cells that showed residual effects of IL-1beta in the presence of IL-1ra, the depolarizing responses were smaller (1.5 ± 0.4 mV) than those observed in the absence of antagonist (2.3 ± 0.1 mV; n = 11). These results suggest that the excitatory effects of IL-1beta on isolated MNCs are mediated specifically by IL-1R1.


Figure 2
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  		Fig. 2. IL-1beta causes an IL-1 receptor type 1 (IL-1R1)-dependent depolarization in MNCs. A: traces show current-clamp recordings of voltage responses from different MNCs (initial voltage near –65 mV) during applications of different concentrations of IL-1beta (bar). B: traces are current-clamp recordings from a single MNC showing the effects of IL-1beta in the presence (open bar, top) and absence of IL-1R1 antagonist (IL-1ra; bottom). Similar results were observed regardless of the order of testing.

 
Ionic basis of IL-1beta actions in MNCs. The ionic basis of the IL-1beta-induced excitation was next examined under voltage clamp. Steady-state current-voltage analysis (Fig. 3A) revealed that the ionic current evoked by IL-1beta had a linear slope between –90 and –20 mV and an average reversal potential (Erev) of –36 ± 2 mV (n = 9). Reducing the concentration of chloride ions in the external solution from 127 to 7 mM did not significantly affect the Erev of the current (–33 ± 1, n = 4; P > 0.05; see Fig. 3B), indicating that the channels modulated by IL-1beta are not permeable to anions. Because no other anions are available to mediate an outward current, and because the value of Erev does not correspond to the equilibrium potential for any of the particular cations in solution, we conclude that the IL-1beta-induced excitation is mediated through the activation of voltage-independent nonselective cation channels.


Figure 3
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  		Fig. 3. Current-voltage (I-V) analysis of the effects of IL-1beta in MNCs. A, left: steady-state I-V relations obtained by applying a slow (15 mV/s) voltage ramp in the absence (control) and presence of 400 nM IL-1beta; right: I-V relation of the IL-1beta-evoked ionic current obtained by subtracting the steady-state I-V relations shown on left. Note that the current is voltage insensitive and that the reversal potential (Erev) lies near –35 mV. B: I-V analysis of the IL-1beta-evoked current recorded in the presence of a low Cl– extracellular solution. Note the similarity of the steady-state (left) and subtracted (right) I-V relations recorded in low Cl– concentration ([Cl–]) solution (7 mM) compared with those recorded in normal (127 mM) [Cl–] solution (A).

 
Effects of IL-1beta on isolated MNCs require intrinsic COX activity. Previous studies of MNCs in hypothalamic slices (10, 18) have indicated that the excitatory actions of IL-1beta in that preparation could be inhibited by Na-salicylate, a general inhibitor of COX. We therefore examined whether Na-salicylate could block IL-1beta-mediated responses in isolated MNCs. In these experiments, 100 µM salicylate was included in all extracellular solutions (including a 15- to 30-min pretreatment) and in solution that filled the recording pipette. Moreover, to exclude the possibility of paracrine effects, drugs were applied using direct perfusion with no other cell lying upstream of the cell being tested. As illustrated in Fig. 4, the progressive increase in membrane conductance evoked by application of 20 nM IL-1beta was dramatically reduced in the presence of salicylate (Fig. 4A). Indeed, the conductance increase measured 120 s following the onset of the application was significantly lower in the presence of salicylate (0.11 ± 0.05 nS, n = 7) compared with that evoked under control conditions (0.56 ± 0.07 nS; n = 16; P = 0.005, Fig. 4B).


Figure 4
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  		Fig. 4. IL-1beta effects are blocked by salicylate. A: graph plots the average time course of mean ± SE changes in membrane conductance observed on adding 20 nM IL-1beta (bar) to the bath in the presence (open circles) and absence (control, solid circles) of 100 µM Na-salicylate. In these experiments, 100 µM salicylate was included in both the external and internal (i.e., recording pipette) solutions. Membrane conductance was monitored from the current response to a –40 mV voltage step applied every 5 s (Vhold = –66 mV). Conductance changes in each cell were calculated by subtracting the average of all control values for that cell from conductance values measured at each time point. B: bar histograms show means ± SE conductance changes observed 2 min following the onset of exposure to IL-1beta for the two groups of cells. *P < 0.05.

 
Effects of IL-1beta involve autocrine production of PGs and EP4 receptors. Previous studies have shown that PGE2 can excite MNCs via the activation of a nonselective cation conductance (10, 30) and that this effect is mediated specifically by the activation of postsynaptic EP4 receptors (28). We therefore examined whether the effects of IL-1beta are due to an autocrine production of PGs by MNCs. Application of 1 µM PGE2 for 120 s under conditions preventing paracrine actions resulted in the activation of a nonselective cation current reversing near –35 mV (n = 3; data not shown). As illustrated in Fig. 5, the increase in membrane conductance induced by PGE2 (1 µM) was significantly inhibited when tested in the presence of 10 µM of the EP4 receptor antagonist AH-23848 (0.11 ± 0.08 nS, n = 8 vs. 0.47 ± 0.09 nS, n = 13 in PGE2 control; P = 0.014). Moreover, AH-23848 significantly inhibited responses induced by direct perfusion of 20 nM IL-1beta (0.04 ± 0.05 nS, n = 6 vs. 0.56 ± 0.07 nS, n = 16 in IL-1beta control; P = 0.003). Finally, when both IL-1beta (20 nM) and PGE2 (1 µM) were applied to MNCs, the effect of IL-beta was occluded by PGE2 (P = 0.227, one-way ANOVA among groups: IL-1beta, PGE2, and IL-beta+PGE2; Fig. 5).


Figure 5
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  		Fig. 5. AH-23848 (AH) blocks the effects of PGE2 and IL-1beta. Bar histograms show the mean ± SE conductance changes observed in many MNCs 2 min following onset of exposure to various drugs. The first pair of bars show that mean increases in conductance caused by 1 µM PGE2 are significantly reduced when tested in the presence of 10 µM AH, an antagonist of the EP4 receptor. *P < 0.05. The second set of bars shows that mean increases in membrane conductance caused by 20 nM IL-1beta are significantly smaller when tested in the presence of 10 µM AH. The effect of IL-beta was occluded by PGE2 when both IL-1beta (20 nM) and PGE2 (1 µM) were applied to MNCs (P = 0.227). *P < 0.005. N.S., no significance.

 
IL-1beta stimulates the osmosensory conductance in MNCs. The cation permeability and voltage independence of the current stimulated by IL-1beta and PGE2 are similar to those of the osmosensory conductance that contributes to osmoreception (20–22, 34) and Na+ detection in MNCs (35). Moreover, previous studies have shown that the neuropeptides ANG II, cholecystokinin, and neurotensin can excite these cells through an upregulation of the osmosensory cation conductance and that the depolarizing effects of these peptides are magnified when elicited under conditions that increase the basal probability of opening of the osmosensing SIC channels (5). We therefore examined the effects of enhancing the background osmosensitive current on the amplitude of the response evoked by IL-1beta. As illustrated in Fig. 6, A and B, the current induced by IL-1beta was significantly enhanced when the volume of the cell was reduced (~15%) by application of negative pressure (–100 mmHg) to the recording pipette (–143 ± 21 pA in the presence of suction vs. –37 ± 5 pA in control; P < 0.05, paired t-test; n = 3). Previous studies have also shown that the osmosensory conductance of MNCs is blocked by Gd3+ with an IC50 near 35 µM (22). We therefore examined the effects of Gd3+ on responses evoked by IL-1beta in 4 MNCs. As shown in Fig. 6, C and D, bath application of 100 µM Gd3+ quickly eliminated the inward current evoked by IL-1beta, as well as the basal inward cation current that was present at rest. Thus, following the addition of IL-1beta, membrane conductance increased from 1.08 ± 0.08 nS to 1.96 ± 0.26 nS (P < 0.05), reflecting the upregulation of osmosensory conductance. On addition of Gd3+, membrane conductance decreased to 0.50 ± 0.17 nS, (n = 4; P < 0.05; one-way ANOVA), consistent with a suppression of both evoked and basal SIC conductance (5, 20–22, 35).


Figure 6
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  		Fig. 6. IL-1beta modulates the osmosensory current in MNCs. Voltage-clamp recordings (Vhold = –66 mV) show current responses to IL-1beta (bars). A: traces show the effects of IL-1beta on the same isolated MNC before (left) and after (right) decreasing cell volume with pipette suction (–100 mmHg). Steady-state inward current caused by the suction-evoked increase in stretch-inhibited cation (SIC) channel activity has been removed to align the baselines of each trace (dashed line) and to emphasize differences in the amplitude of the response to IL-1beta in the two conditions. Note that the increase in current noise reflects the activation of SIC channels. Large vertical transients are current responses caused by the voltage ramps used for I-V analysis (not shown). B: means ± SE amplitude of the IL-1beta evoked current recorded at –66 mV in the absence (before suction) and presence (after suction) of pipette suction (n = 3; *P < 0.05; paired Student's t-test). C: current trace showing that the inward current evoked by IL-1beta (bar) can be blocked by bath application of Gd3+ (open bar). The net outward current observed in Gd3+ compared with control baseline (dotted line) is due to the blockade of all SIC channels, including those already open at rest. D: means ± SE membrane conductance recorded in the presence of IL-1beta before (IL-1beta; n = 4) and after (IL-1beta + Gd) addition of 100 µM Gd3+ to the bath. *P < 0.05 (one-way ANOVA).

 
IL-1beta does not affect the volume or mechanosensitivity of MNCs. Because the SIC conductance of MNCs is mechanically activated during decreases in cell volume, the upregulation of this conductance in the presence of IL-1beta might be due to a drug-evoked decrease in cell volume. However, application of 20 nM IL-1beta alone did not provoke significant changes in cell volume (volume at 120 s was 99.98 ± 0.004% of control; P = 0.97; n = 7). A previous study has indicated that the osmoresponsiveness of the neurohypophyseal axis is increased during endotoxinemia (12). Thus it is possible that the intrinsic osmosensitivity of MNCs is increased in the presence of IL-1beta, even if the cytokine does not itself provoke a decrease in cell volume. Because the osmosensitivity of MNCs is due to the mechanical regulation of the SIC conductance during osmotically evoked changes in cell volume, we examined whether IL-1beta could increase the mechanosensitivity of MNCs. Cells were maintained at a holding potential of –66 mV, and hyperpolarizing steps to –106 mV were applied every 5 s to monitor membrane conductance. Images were captured simultaneously to monitor changes in cell volume. As illustrated in Fig. 7A, reducing pipette pressure for 60 s caused a progressive decrease in cell volume accompanied by the emergence of an inward current and an increase in membrane conductance. This experiment was repeated in eight MNCs under control conditions, and in eight MNCs exposed to 20 nM IL-1beta. As shown in Fig. 7B, no significant differences were noted in the mean amplitudes of the suction-evoked volume decrease (–16.3 ± 0.9% control vs. –14.3 ± 0.9% IL-1beta; P = 0.141) or in the conductance increase (+0.42 ± 0.17 nS control vs. +0.32 ± 0.20 nS IL-1beta; P = 0.72), observed in the two groups of cells. Moreover, the mean MI (conductance change/normalized volume change) was not different in control (2.6 ± 1.1 pS/n{Delta}V) and IL-1beta-treated cells (2.3 ± 1.5 pS/n{Delta}V; P = 0.61).


Figure 7
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  		Fig. 7. IL-1beta does not affect the mechanosensitivity of MNCs. A, top trace: percent changes in cell volume ({Delta}V; relative to control) evoked in an isolated MNC by applying suction (bar; –100 mmHg) through a recording pipette. Middle trace: corresponding changes in whole cell current (thick part of trace; Vhold = –66 mV) and current responses ({Delta}I, downward deflections) to voltage steps (–40 mV; 500 ms). Bottom trace: changes in membrane conductance ({Delta}G) as a function of time during the same experiment. B: bar graphs show means ± SE decreases in cell volume, changes in conductance ({Delta}G) and mechanosensitivity (MI) observed at 60 s in MNCs tested under control conditions (control; n = 8) and in 8 MNCs that had been exposed to 20 nM IL-1beta for 120 s before suction was applied (n = 8).

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Hypothalamic expression of IL-1beta increases rapidly following systemic infection (23, 26, 33), and the central and peripheral release of IL-1beta that occurs under these conditions is believed to affect neuronal activity in a manner that orchestrates the behavioral and neuroendocrine responses that contribute to host defense (e.g., 1, 2, 9, 13, 16, 17, 25, 32). The release of vasopressin from the neurohypophysis, in particular, is an important adaptive response that contributes to the maintenance of tissue perfusion during the early stage of septic shock (13), and much evidence supports the hypothesis that IL-1beta may contribute to the increase in vasopressin release that occurs under such conditions. Indeed, intracerebroventricular infusions of IL-1beta provoke the release of vasopressin in vivo (16, 31, 36), and bath application of IL-1beta can stimulate vasopressin release from hypothalamic explants in vitro (37). Moreover, bath application of exogenous IL-1beta has been shown to excite MNCs in hypothalamic slices (10), a response that is not blocked by tetrodotoxin (10, 18) and that appears to be mediated through the activation of a nonselective cation conductance (10). Thus IL-1beta may provoke vasopressin release by increasing the electrical activity of MNCs through effects that take place on or near the somata of these hypothalamic neurons. Interestingly, the excitatory effects of IL-1beta on MNCs in hypothalamic slices are antagonized by COX inhibitors (10, 18), indicating that synthesis and release of PGs is required to mediate the effects of IL-1R1 activation. Further support for this hypothesis has come from the observation that EP4 receptors are expressed in the supraoptic nucleus (38) and that PGE2 can excite acutely isolated MNCs by increasing a nonselective cation conductance (30) upon activation of postsynaptic EP4 receptors (28). Although these observations suggest that IL-1R1-mediated PG release may excite MNCs through an effect involving postsynaptic EP4 receptors, they do not indicate whether MNCs respond directly to IL-1beta or whether the effects of the cytokine are mediated by PGs released from neighboring IL-1R1 expressing cells. Indeed, the observation that tetrodotoxin does not prevent the depolarizing effects of IL-1beta on MNCs in slices (10, 18) indicates that the effects of the cytokine are not mediated by activity-dependent exocytotic release of another transmitter. However, it does not exclude the possibility that a diffusible agent, such as PGE2, might be released from other cells and excite MNCs through a paracrine action.

MNCs are directly excited by IL-1beta. Application of IL-1beta caused a potent excitation of MNCs, an effect that could be blocked by IL-1ra, an antagonist of the IL-1R1 (1, 26). This response was mediated via the intrinsic activation of a nonselective cation conductance under conditions excluding the possibility of paracrine effects, indicating that individual MNCs contain all of the cellular machinery required to transduce the effects of IL-1beta. Although we did not identify the neurons recorded as vasopressin- or oxytocin-containing in our experiments, these cells respectively account for 60 and 40% of the MNCs isolated by our procedure (11). Because all cells tested with suprathreshold (>20 pM) concentrations of IL-1beta were responsive to the drug, it is likely that both oxytocin- and vasopressin-releasing MNCs express IL-1R1, as well as other signaling elements required for cellular activation. Indeed, previous studies have indicated that IL-1beta can modulate the release of both oxytocin and vasopressin (29).

Effects of IL-1beta involve an EP4 receptor-mediated autocrine effect. The effects of IL-1beta observed during direct perfusion experiments were blocked in the presence of sodium salicylate, confirming a requirement for COX activity and PG synthesis in the genesis of the IL-1beta response. Because the possibility of a paracrine effect was eliminated in our experiments, any PG produced by the neuron being tested would have to activate intrinsic receptors to mediate a response. In agreement with this hypothesis, direct application of PGE2 to a single neuron also caused the activation of a nonselective cation conductance. Moreover, this effect could be blocked by AH-23848, indicating the involvement of intrinsic EP4 receptors. Finally, if PG synthesis and EP4 receptors act as intermediates in the mediation of IL-1beta effects on MNCs, then the direct effects of the cytokine should be inhibited by EP4 receptor blockade and occluded by prior activation of such receptors. Indeed, the effects of IL-1beta were abolished in the presence of AH-23848 and occluded in the presence of PGE2. Although our results reveal the existence of an autocrine PGE2-mediated response to direct IL-1R1 activation in MNCs, they do not rule out the possible involvement of paracrine effects. Indeed, previous studies have shown that PGE2 can also excite MNCs through a presynaptic inhibition of GABA release mediated by EP3 receptors (28), and a recent study has shown that excitatory synaptic transmission in the hippocampus can be enhanced retrogradely via the activation of presynaptic EP2 receptors in response to postsynaptically synthesized PGE2 (27). Whether IL-1R1-mediated PGE2 release by MNC somata can retrogradely modulate synaptic transmission remains to be determined.

IL-1beta upregulates the osmosensory conductance of MNCs. In a recent report, Grinevich et al. (12) showed that intraperitoneal injection of the bacterial endotoxin lipopolysaccharide causes an increase in vasopressin release that is associated with an increase in the osmoresponsiveness of the hypothalamo-neurohypophysial system. Because the effects of lipopolysaccharide are mediated, in part, by central actions of IL-1beta (e.g., 9), it is reasonable to hypothesize that the effects of IL-1beta might be mediated via actions on one of the mechanisms involved in the osmotic regulation of MNCs (14, 34). Voltage-clamp analysis showed that the excitatory effects of IL-1beta on isolated MNCs were due to the activation of a Gd3+-sensitive, voltage-insensitive, nonselective cation current that reverses near –35 mV. These properties are similar to those of the SIC channels that contribute to osmoreception (20–22) and sodium detection (35) in MNCs. Moreover, the amplitude of the current evoked by IL-1beta was enhanced under conditions that increase the opening probability of SIC channels, as previously shown for ANG II, cholecystokinin, and neurotensin (5). Because these channels are mechanically regulated, we also explored the possibility that SIC conductance activation might have been due to an IL-1beta-mediated decrease in cell volume or to a change in mechanosensitivity. Our data showed that IL-1beta affects neither the volume nor mechanosensitivity of MNCs. Although our assessment of mechanosensitivity was based on a stimulus that yielded a maximal change in volume of about –15%, it is important to note that the MI computed by our method also applied to the smallest volume changes detectable (see EXPERIMENTAL PROCEDURES). Thus IL-1beta is unlikely to affect the activation of SIC channels by physiologically relevant hypertonic stimuli. Although additional studies will be required to define the mechanism by which IL-1beta and PGE2 activate the nonselective cation current in MNCs, our observations suggest that this response is mediated specifically by the activation of SIC channels.

Possible functional role of intrinsic IL-1R1 activation in MNCs. Although the mechanical coupling between changes in cell volume and SIC channel activity is not altered by IL-1beta, the activation of the SIC conductance during IL-1R1 activation will mediate an inward current and, consequently, a depolarization of the membrane potential (10, 18). This effect will promote an increase in the rate of action potential discharge from MNCs and thus enhance the release of vasopressin from axon terminals in the neurohypophysis (24). Moreover, by increasing the overall fraction of excitatory postsynaptic potentials that cross spike threshold, the IL-1beta-mediated depolarization might increase the excitatory impact of the glutamatergic projection from osmosensitive organum vasculosum lamina terminalis neurons to MNCs in the supraoptic nucleus (4) and thereby the osmoresponsiveness of the neurohypophysial system (3, 34). Further work will be required to confirm this hypothesis.

In conclusion, our experiments reveal that neurons in the supraoptic nucleus, like those in the subfornical organ (7), appear to express all of the molecular machinery required for IL-1R1-mediated transduction of depolarizing responses to IL-1beta. In particular, our results show that the excitatory effects of this cytokine require the COX-dependent synthesis of a PG, and an upregulation of nonselective cation conductance that depends on the autocrine activation of EP4 receptors. The mechanism by which PG upregulates the cation conductance of MNCs remains to be determined.


   ACKNOWLEDGMENTS
 
This work was supported by Canadian Institutes of Health Research Senior Investigator and Operating awards, and by a James McGill Research Chair (to C. W. Bourque). Z. Zhang was supported by Studentship awards from the Research Institute of the McGill University Health Center and the Heart and Stroke Foundation of Canada.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Bourque, Rm. L7–216, Division of Neurology, Montreal General Hospital, 1650 Cedar Ave., Montreal, QC, Canada, H3G 1A4 (E-mail: charles.bourque{at}mcgill.ca)

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.

* Y. Chakfe and Z. Zhang contributed equally to this paper. Back


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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