A novel osmosensitive voltage gated cation current in rat supraoptic neurones
1 Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E5
Abstract
The magnocellular neurosecretory cells of the hypothalamus (MNCs) regulate water balance by releasing vasopressin and oxytocin as a function of plasma osmolality. Release is determined largely by the rate and pattern of action potentials generated in the MNC somata. Changes in firing are mediated in part by a stretch-inactivated non-selective cation current that causes the cells to depolarize when increased osmolality leads to cell shrinkage. We have obtained evidence for a new current that may regulate MNC firing during changes in external osmolality, using whole-cell patch clamp of acutely isolated rat MNC somata. In internal and external solutions lacking K+, with high concentrations of TEA, and with Na+ as the only likely permeant cation, the current appears as a slow inward current during depolarizations and yields a large tail current upon return to the holding potential of –80 mV. Approximately 60% of the MNCs tested (79 out of 134 cells) displayed a large increase in tail current density (from 5.2 ± 0.9 to 10.5 ± 1.4 pA pF–1; P < 0.001) following an increase in external osmolality from 295 to 325 mosmol kg–1. The current is activated by depolarization to potentials above –60 mV and does not appear to depend on changes in internal Ca2+. The current is carried by Na+ under these conditions, but is blocked by Cs+ and Ba2+ and by internal K+, which suggests that the current could be a K+ current under physiological conditions. This current could play an important role in regulating the response of MNCs to osmolality.
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Introduction
The magnocellular neurosecretory cells of the hypothalamus (MNCs) are osmoreceptors (Bourque & Oliet, 1997). These cells sense changes in plasma osmolality and act to maintain osmotic homeostasis by regulating the systemic release of vasopressin (VP), which controls water excretion from the kidneys, and oxytocin (OT), which acts as a natiuretic hormone as well as regulating lactation and uterine contractions at parturition (Poulain & Wakerley, 1982; Bourque & Oliet, 1997). VP and OT are synthesized in the somata of the MNCs, which are located primarily in the supraoptic nucleus (SON) and paraventricular nucleus of the hypothalamus, and released into the circulation from the MNC terminals in the neurohypophysis following Ca2+ influx through voltage gated Ca2+ channels (Fisher & Bourque, 2001). The amount of release is primarily determined by the rate and pattern of action potentials generated in the MNC somata (Bicknell, 1988). Both VP- and OT-ergic MNCs fire irregularly and infrequently when plasma osmolality is near normal, and both fire more rapidly as the external osmolality increases (Poulain & Wakerley, 1982; Bourque & Oliet, 1997). VP-ergic MNCs also respond to increased osmolality by adopting a phasic pattern of firing, which is characterized by bursts of action potentials lasting tens of seconds followed by rest intervals of about the same length (Poulain & Wakerley, 1982). Most OT MNCs do not adopt a phasic pattern of firing in response to elevations of osmolality, but do fire rapid bursts during lactation and at parturition (Poulain & Wakerley, 1982). The adoption of a phasic pattern of firing is important physiologically because this maximizes hormone release (Bicknell, 1988).
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Increases in firing frequency involve an increase in excitatory drive from other osmosensitive cells (Richard & Bourque, 1995), and a continuous excitatory drive is required to sustain phasic firing (Brown et al. 2004). Firing patterns are also influenced by release of local modulators such as VP, which is released in an autocrine fashion from the somatodendritic region of the MNCs (Ludwig & Pittman, 2003), and peptides that are coreleased with VP, such as eprin (De Mota et al. 2004) and dynorphin (Brown & Bourque, 2004). Taurine, which exerts an inhibitory influence on MNCs, is released from glia in the SON in a fashion inversely proportional to osmolality (Hussy et al. 1997). Osmotically regulated changes in MNC firing patterns, however, are shaped by the interplay of osmosensitive and activity-dependent ionic currents in the MNCs. Increases in MNC responsiveness depend in large part on a depolarization of the MNC plasma membrane mediated by a stretch-inactivated cation channel or SIC (Oliet & Bourque, 1993). Since the MNCs do not display regulatory volume changes in response to osmotic swelling or shrinkage (Zhang & Bourque, 2003), increases in osmolality lead to a sustained depolarization of the MNC membrane, an increased likelihood of cell firing in response to excitatory postsynaptic potentials, and an increase in hormone release. Initiation of phasic firing requires a depolarizing afterpotential (DAP) that follows evoked action potentials and can summate into the plateau potentials required for phasic firing in MNCs (Andrew & Dudek, 1983). The DAP may result from Ca2+-dependent activation of a non-selective cation current (Ghamari-Langroudi & Bourque, 1998, 2002) and/or the Ca2+-dependent inactivation of a K+ current (Li & Hatton, 1997). The MNCs also express hyperpolarizing afterpotentials, which are mediated by K+ currents (Bourque & Brown, 1987; Greffrath et al. 1998; Ghamari-Langroudi & Bourque, 2004) and could contribute to the cessation of firing required for burst termination. Termination of bursts may also involve a progressive decrease in the amplitude of the DAP and plateau potential caused by the auto-inhibitory actions of dynorphin released from MNC somata during bursts (Brown & Bourque, 2004).
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The acceleration of MNC firing that occurs as osmolality increases, and the transition that occurs between rapid continuous and phasic firing (Bourque & Renaud, 1984), may involve modulation of ion channels other than the SIC. We have therefore sought to determine whether other currents expressed in the MNCs are sensitive to changes in osmolality. We report here a slowly activating voltage gated cation current that increases in amplitude in response to increases in external osmolality. The properties of this current suggest that it could contribute to an afterpotential that regulates MNC firing patterns. This current might therefore play an important role in the regulation of VP and OT release. Some of these results have been presented in abstract form (Liu & Fisher, 2003).
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Methods
Animals and cell preparation
Male Long-Evans rats (200–300 g) were anaesthetized with halothane and killed by decapitation according to a protocol approved by the University of Saskatchewan Animal Care Committee. MNCs were isolated as previously described (Oliet & Bourque, 1992; Fisher & Bourque, 1995). The brain was removed and blocks of tissue containing most of the two supraoptic nuclei were obtained. The tissue blocks were incubated for 90 min at 34°C with an oxygenated (100% O2) Pipes solution (pH 7.1) composed of (mM): NaCl, 120; KCl, 5; MgCl2, 1; CaCl2, 1; Pipes, 20; glucose, 25; and containing trypsin (Type XI, 0.6 mg ml–1). The tissues were then transferred into oxygenated Pipes solution without trypsin for 30–120 min at room temperature. The tissues were gently triturated with fire-polished pipettes to disperse the cells onto glass-bottomed recording dishes for use the same day.
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Electrophysiological recording
Whole-cell patch-clamp recordings were made using fire-polished micropipettes (A-M Systems Inc., Carlsborg, WA, USA) prepared using a P-97 horizontal pipette puller (Sutter Instrument Co., Novato, CA, USA) and polished using a microforge (Narishige, Tokyo, Japan). Currents were recorded at room temperature using an EPC-9 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) operated with Pulse software (HEKA), and were low pass filtered (2 kHz). TEA/Na+ external solution (pH 7.35) was composed of (mM): NaCl, 120; CaCl2, 2; glucose, 10; Hepes, 10; TTX, 0.0002; TEA, 20; and 4-AP, 4 (Fisher & Bourque, 1995). The recording electrodes (2.5–5 M) were filled with a solution (pH 7.2) containing (mM): Tris, 110; MgCl2, 1; EGTA, 1.6; TEA, 40; Na2ATP, 2; and phosphocreatine, 14. In some experiments, external Na+ was replaced with N-methyl-D-glucamine (NMDG, 90 mM). The osmolality of the solutions were measured using a Vapro pressure osmometer (Wescor Inc., Logan, UT, USA) and adjusted to the desired osmolality by adding mannitol as required. All chemicals were from Sigma (St Louis, MO, USA).
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Data analysis
Tail current amplitudes were measured by subtracting the amplitude after the current was fully depolarized from that 20 ms after the end of the depolarization pulse. The current amplitude was divided by the cell capacitance to normalize for cell size in most experiments. Cell capacitance was measured automatically by the c-slow compensation function of the Pulse software. The mean capacitance of the responding MNCs was 12.5 ± 0.9 pF, which is close to that previously observed for a selected group of MNCs (15.2 ± 0.4 pF; Oliet & Bourque, 1992). Data are shown as the mean ± S.E.M. Statistical significance was estimated by Student's paired or unpaired t test as described. A P-value of less than 0.05 was regarded as significant.
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Results
Isolation of the osmosensitive cation current (OC)
When acutely isolated MNCs were patch clamped in the whole-cell mode using TEA/Na+ external solution, depolarizing steps were followed by a long lasting tail current (Fig. 1A). These solutions contained TTX to block Na+ currents, and K+ currents were diminished by the presence of 4-AP in the external solution, TEA in internal and external solutions, and by the absence of internal and external K+. Since ICa in MNCs has been shown to be fully deactivated within a few milliseconds (Foehring & Armstrong, 1996; Harayama et al. 1998), this tail current (which deactivated over about 400 ms) appeared to result from a current other than Ca2+ current. Depolarization from a holding potential of –80 mV to 0 mV for 400 ms resulted in a tail current with an amplitude greater than 20 pA (and as high as 400 pA) in 145 out of 175 MNCs. The amplitude of this tail current was sensitive to changes in the osmolality of the external solution (Fig. 1A and B). When the external osmolality was increased from 295 to 325 mosmol kg–1, a large increase in the amplitude of the inward tail current was frequently observed, as well as a small increase in inward current during the depolarizing step (Fig. 1A, left). Overall, about 60% of the cells (79 out of 134 cells) underwent a marked increase in tail current amplitude; the mean tail current density in these sensitive cells doubled from 5.2 ± 0.9 pA pF–1 in 295 mosmol kg–1 to 10.5 ± 1.4 pA pF–1 in 325 mosmol kg–1 (P < 0.001; Fig. 1A, right). Curve fitting of the tail current in 325 mosmol kg–1 showed that the current deactivated with a time constant of 75.0 ± 9.1 ms. The change in amplitude was reversible when the external solution was returned to 295 mosmol kg–1 (data not shown). Furthermore, the amplitude of this tail current decreased when the osmolality of the external solution was decreased from normal (295 mosmol kg–1) to hypotonic (265 mosmol kg–1; Fig. 1B). Figure 1B (left) shows the amplitudes of currents evoked in a single MNC in 295, 265 and 325 mosmol kg–1; Fig. 1B (right) shows the time course of these changes in the same cell. Similar results were seen in 8 out of 15 cells tested. Since the osmolality was changed using a gravity-fed perfusion system, it is likely that the rate of change in current amplitude was determined largely by the perfusion rate. We will henceforth refer to the current whose amplitude is changed with changes in external osmolality as the osmosensitive current or OC. The current required to hold the cells at –80 mV was unchanged by changes in the external osmolality, which suggests that the SIC is blocked under these conditions (probably by the presence of internal TEA; authors' unpublished observations).
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A, left, currents evoked by depolarizing an MNC from –80 mV to 0 mV in a 295 mosmol kg–1 solution and then in a 325 mosmol kg–1 solution. Note the large increase in the amplitude of the tail current. Right, the mean tail current density in 79 responding cells before and after increasing the osmolality of the external solution from 295 to 325 mosmol kg–1 (P < 0.001). B, left, currents evoked by depolarizing steps in an MNC in 295, 265 and 325 mosmol kg–1 solutions. Right, the time course of changes in tail current amplitude (normalized to the maximum evoked amplitude) during the noted changes in external osmolality.
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We tested the ionic basis of the OC by replacing external Na+ with NMDG. Figure 2A shows that the OC density in 325 mosmol kg–1 TEA/Na+ solution (12.5 ± 3.676 pA pF–1) was nearly abolished after Na+ was replaced with NMDG (0.522 ± 0.481 pA pF–1; n = 6). There was also a small decrease in inward current during the depolarizing step. These data support the conclusion that the OC is a cation current that increases in amplitude as the external osmolality is increased, and that Na+ is the major charge carrier under these conditions. In support of this conclusion, including 40 mM Na+ in the internal solution (by replacing 40 mM Tris) caused a significant decrease in the amplitude of the tail current (to 6.8 ± 1.0 pA pF–1 at –80 mV, n = 8, compared to 10.5 ± 1.4 pA pF–1 as shown in Fig. 1), presumably because the driving force for Na+ was decreased.
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A, left, currents evoked by depolarizing an MNC from –80 mV to 0 mV in a 325 mosmol kg–1 TEA/Na+ solution and after replacing Na+ with NMDG. Note that the tail current amplitude is dramatically decreased and that there is a small decrease in the inward current evoked during the step. Right, the mean tail current density in 6 cells before and after replacing Na+ with NMDG (P < 0.001). B, left, currents evoked by depolarizing an MNC from –80 mV to 0 mV in 295 mosmol kg–1 and 325 mosmol kg–1 TEA/NMDG solutions. There was no change in the amplitude of evoked Ca2+ current or tail current. Right, the mean Ca2+ current density in 5 cells in 295 and 325 mosmol kg–1 TEA/NMDG solutions. C, left, currents evoked in 295 and 325 mosmol kg–1 solutions with 5 mM BAPTA included in the internal solution. Right, tail current density in 5 cells in 295 and 325 mosmol kg–1 solutions with 5 mM BAPTA included in the patch pipette.
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To test whether Ca2+ currents are sensitive to changes in osmolality, Ca2+ currents were evoked in TEA/NMDG external solution. Under these conditions, there was no change in amplitude of either the Ca2+ current or the tail currents that followed (Fig. 2B). The Ca2+ currents in MNCs therefore do not appear to be osmosensitive.
To test whether the OC is activated by an increase in intracellular Ca2+, we activated the OC in the presence of BAPTA in the pipette solution. BAPTA caused no discernable difference in the amplitude of the OC or its increase in amplitude in response to increased osmolality (Fig. 2C). These data suggest that the OC is not dependent on increases in intracellular Ca2+ for activation and that it is primarily voltage dependent.
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Kinetics and Ca2+ dependence of the OC
Figure 3A shows that the amplitude of the tail current is time dependent and increases as the length of depolarizing steps increases over a range from 10 to 500 ms. The traces above show currents evoked by steps from –80 mV to 0 mV of the indicated lengths and the plot shows the mean results obtained from 15 cells. The tail current was fully activated by depolarizing pulses of about 400 ms (Fig. 3A). The OC therefore activates and deactivates over hundreds of milliseconds.
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A, tail current density following depolarization of different lengths. Cells in 325 mosmol kg–1 TEA/Na+ solution were stepped from a holding potential of –80 mV to 0 mV for different lengths of time (from 10 ms to 500 ms) and then returned to –80 mV. The traces above show the currents evoked by these steps and the plot below shows the mean results from 15 cells. B, tail current density evoked by steps to different voltages. Cells were stepped from a holding potential of –80 mV to a series of potentials between –70 mV and +20 mV for 400 ms (in 10 mV increments every 1.5 s) and then returned to –80 mV in 295 and then 325 mosmol kg–1 TEA/Na+ solution. The traces above show families of tail currents (commencing 20 ms after the depolarizing step) evoked in the same cell in 295 and 325 mosmol kg–1 solutions. The plot below shows the mean current–voltage relationship for 9 cells. The increase in current caused by the change to 325 mosmol kg–1 solution is plotted using triangles.
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Figure 3B plots the mean current–voltage relationships for activation of the tail current in 295 and 325 mosmol kg–1 solutions in nine MNCs. The OC was activated by steps to potentials greater than –60 mV and fully activated at 0 or +10 mV. These data support the conclusion that OC activation is primarily dependent on voltage, rather than Ca2+ influx, since the OC is activated at potentials at which there is no measurable Ca2+ current under these conditions, and does not appear to decrease in amplitude at potentials at which the Ca2+ current has decreased in amplitude (Ca2+ current peaks at –10 mV in MNCs and declines at higher potentials (Fisher & Bourque, 1995). Furthermore, OC amplitude does not correlate well with the amplitude of Ca2+ current in individual MNCs, and is stable over time despite the rundown of Ca2+ current (data not shown).
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Pharmacological properties
The amplitude of the OC was reversibly inhibited by the addition of 1 mM Cs+ in 325 mosmol kg–1 TEA/Na+ external solution (Fig. 4; n = 18). The traces in Fig. 4A (left) show the tail current before, during, and after the addition of Cs+. Ca2+ currents did not appear to be affected. Figure 4A (right) shows the tail current density before (7.572 ± 5.957 pA pF–1) and after (0.882 ± 0.7015 pA pF–1) addition of Cs+, indicating an 88% decrease in amplitude. When Cs+ (10 mM) was included in the internal solution, the tail current in amplitude was very low in 295 mosmol kg–1 solution (1.48 ± 0.35 pA pF–1, P > 0.05) and was not significantly increased by perfusion with 325 mosmol kg–1 solution (1.79 ± 0.31 pA pF–1) in four cells (Fig. 4B). The OC is therefore sensitive to both external and internal Cs+. The inclusion of Cs+ in internal solutions used in previous studies of voltage gated Ca2+ currents in MNCs (Foehring & Armstrong, 1996; Harayama et al. 1998) may help to explain why this tail current was not observed.
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A, left, currents evoked by depolarizing an MNC from –80 mV to 0 mV in 325 mosmol kg–1 solutions before, during and after addition of 1 mM Cs+. Right, the mean tail current density of 18 cells before and after addition of 1 mM Cs+ (P < 0.001). B, left, currents evoked in 295 and 325 mosmol kg–1 solutions with 10 mM Cs+ included in the internal solution. Right, the mean tail current density of 4 cells in 295 and 325 mosmol kg–1 solutions with 10 mM Cs+ included in the internal solution (P > 0.05). C, left, currents evoked in 325 mosmol kg–1 external solution before, during and after addition of 0.25 mM Ba2+. Right, the mean tail current density of 9 cells before and after addition of 0.25 mM Ba2+ (P < 0.001). D, left, currents evoked in an MNC with an internal solution containing 5 mM K+ in 295, 325 and 325 mosmol kg–1 external solution in which 40 mM Na+ was replaced with 40 mM K+. Right, the mean tail current densities in MNCs recorded with an internal solution containing 5 mM K+ in 295, 325 and 325 mosmol kg–1 external solution containing 40 mM K+ (n = 11). The addition of 40 mM K+ caused a significant increase in tail current density (P < 0.001).
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The traces in Fig. 4C (left), show the currents evoked before, during, and after the addition of Ba2+ in 325 mosmol kg–1 external solution. Figure 4C (right) shows the mean tail current density before and after application of Ba2+. The OC amplitude was reversibly blocked from 7.81 ± 1.69 pA pF–1 to 0.996 ± 0.104 pA pF–1 by 250 μM Ba2+ in nine MNCs (Fig. 4C, right). This represents an 89% blockade. These results indicate that the OC is sensitive to external Ba2+.
, 百拇医药 The block of the OC by Ba2+ and Cs+ suggests the possibility that the OC could be a K+ current, whose ion selectivity is altered in the absence of K+ such that Na+ becomes permeant. In support of this hypothesis is the observation (Fig. 4D) that the addition of a small concentration of K+ (5 mM) to the internal solution results in a decreased tail current density in 295 mosmol kg–1 solution (0.73 ± 0.27 pA pF–1) and prevents the increase in current when the solution is changed to 325 mosmol kg–1 (0.86 ± 0.26 pA pF–1; n = 11). Furthermore, addition of 40 mM K+ to the 325 mosmol kg–1 external solution in exchange for 40 mM Na+ leads to the appearance of an inward tail current (5.24 ± 0.45 pA pF–1; n = 11). These data support the hypothesis that the OC is a K+ current.
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Discussion
The identification in the somata of MNCs of an osmosensitive, voltage gated, slowly activating cation current is a novel finding that could have important implications in the regulation of MNC firing and neuropeptide release. The block of the OC by Cs+ and Ba2+, and the decrease of Na+ permeability by the inclusion of K+ in the internal solution suggest that the OC is most likely to be a K+ current. Our data suggest that the OC is clearly different from the SIC, which is a mechanosensitive non-selective cation channel that is relatively insensitive to voltage (Oliet & Bourque, 1993) and which is not sensitive to Cs+ (C. W. Bourque, personal communication). The OC is also clearly distinct from a stretch-sensitive voltage-insensitive K+ leak channel that has been identified using single channel cell-attached recordings of MNC somata (Han et al. 2003). The OC, in contrast to both of these currents, appears to be a voltage gated channel whose amplitude is modulated by changes in the external osmolality. It remains to be seen whether this effect is mediated by a direct action of membrane tension on the channel, or is mediated via a second messenger system that is itself osmosensitive.
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Our observation that only about 60% of the MNCs expressed currents that responded to changes in osmolality is unexplained. Since VP and OT MNCs respond differently to osmotic stimulation, it is possible that a greater proportion of one type expresses the OC. Whole-cell patch- clamp studies of MNCs in slices have demonstrated, however, that MNCs can be converted from phasic to continuous firing and vice versa by altering internal Ca2+ buffering (Li et al. 1995). These data suggest that all MNCs express the currents necessary to support phasic firing and that differences in firing patterns between OT and VP MNCs result in part from differences in the levels of Ca2+ binding proteins. The distribution of the OC in VP and OT MNCs will therefore need to be determined.
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Although the physiological role of the OC is not yet clear, it seems reasonable to speculate that it is involved in mediating the changes in MNC firing that occur in response to changes in external osmolality. If the OC is a Na+ current or a non-selective cation current, its activation could contribute to an increase in the size of the DAP and/or the plateau potential during hyperosmolality. Increases in osmolality, however, do not appear to change the amplitude of the DAP (Bourque, 1989). If the OC is a K+ current, it might contribute to the afterhyperpolarizations (AHPs) observed in MNCs. These include a medium AHP (mAHP) lasting 200–500 ms (Bourque & Brown, 1987; Armstrong et al. 1994; Kirkpatrick & Bourque, 1996), and a slow AHP (sAHP), which decays over seconds (Greffrath et al. 1998). Both of these currents are important in regulating firing patterns. Blockade of the mAHP with apamin causes a decrease in burst length (Kirkpatrick & Bourque, 1996) and inhibition of the sAHP by muscarinic agonists increases burst length (Ghamari-Langroudi & Bourque, 2004). Both the mAHP and the sAHP are selectively elevated in OT MNCs during lactation (Teruyama & Armstrong, 2005), suggesting that an increase in AHP amplitude may be an adaptation that favours burst firing. Although both these currents are largely Ca2+ dependent, it is possible that the OC could contribute to the mAHP and/or the sAHP, and that an increase in its amplitude during increases in external osmolality might influence firing patterns. Future studies will pursue elucidation of the ionic selectivity and molecular nature of the OC, the molecular mechanism underlying its osmosensitivity, and its physiological role.
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Abstract
The magnocellular neurosecretory cells of the hypothalamus (MNCs) regulate water balance by releasing vasopressin and oxytocin as a function of plasma osmolality. Release is determined largely by the rate and pattern of action potentials generated in the MNC somata. Changes in firing are mediated in part by a stretch-inactivated non-selective cation current that causes the cells to depolarize when increased osmolality leads to cell shrinkage. We have obtained evidence for a new current that may regulate MNC firing during changes in external osmolality, using whole-cell patch clamp of acutely isolated rat MNC somata. In internal and external solutions lacking K+, with high concentrations of TEA, and with Na+ as the only likely permeant cation, the current appears as a slow inward current during depolarizations and yields a large tail current upon return to the holding potential of –80 mV. Approximately 60% of the MNCs tested (79 out of 134 cells) displayed a large increase in tail current density (from 5.2 ± 0.9 to 10.5 ± 1.4 pA pF–1; P < 0.001) following an increase in external osmolality from 295 to 325 mosmol kg–1. The current is activated by depolarization to potentials above –60 mV and does not appear to depend on changes in internal Ca2+. The current is carried by Na+ under these conditions, but is blocked by Cs+ and Ba2+ and by internal K+, which suggests that the current could be a K+ current under physiological conditions. This current could play an important role in regulating the response of MNCs to osmolality.
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Introduction
The magnocellular neurosecretory cells of the hypothalamus (MNCs) are osmoreceptors (Bourque & Oliet, 1997). These cells sense changes in plasma osmolality and act to maintain osmotic homeostasis by regulating the systemic release of vasopressin (VP), which controls water excretion from the kidneys, and oxytocin (OT), which acts as a natiuretic hormone as well as regulating lactation and uterine contractions at parturition (Poulain & Wakerley, 1982; Bourque & Oliet, 1997). VP and OT are synthesized in the somata of the MNCs, which are located primarily in the supraoptic nucleus (SON) and paraventricular nucleus of the hypothalamus, and released into the circulation from the MNC terminals in the neurohypophysis following Ca2+ influx through voltage gated Ca2+ channels (Fisher & Bourque, 2001). The amount of release is primarily determined by the rate and pattern of action potentials generated in the MNC somata (Bicknell, 1988). Both VP- and OT-ergic MNCs fire irregularly and infrequently when plasma osmolality is near normal, and both fire more rapidly as the external osmolality increases (Poulain & Wakerley, 1982; Bourque & Oliet, 1997). VP-ergic MNCs also respond to increased osmolality by adopting a phasic pattern of firing, which is characterized by bursts of action potentials lasting tens of seconds followed by rest intervals of about the same length (Poulain & Wakerley, 1982). Most OT MNCs do not adopt a phasic pattern of firing in response to elevations of osmolality, but do fire rapid bursts during lactation and at parturition (Poulain & Wakerley, 1982). The adoption of a phasic pattern of firing is important physiologically because this maximizes hormone release (Bicknell, 1988).
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Increases in firing frequency involve an increase in excitatory drive from other osmosensitive cells (Richard & Bourque, 1995), and a continuous excitatory drive is required to sustain phasic firing (Brown et al. 2004). Firing patterns are also influenced by release of local modulators such as VP, which is released in an autocrine fashion from the somatodendritic region of the MNCs (Ludwig & Pittman, 2003), and peptides that are coreleased with VP, such as eprin (De Mota et al. 2004) and dynorphin (Brown & Bourque, 2004). Taurine, which exerts an inhibitory influence on MNCs, is released from glia in the SON in a fashion inversely proportional to osmolality (Hussy et al. 1997). Osmotically regulated changes in MNC firing patterns, however, are shaped by the interplay of osmosensitive and activity-dependent ionic currents in the MNCs. Increases in MNC responsiveness depend in large part on a depolarization of the MNC plasma membrane mediated by a stretch-inactivated cation channel or SIC (Oliet & Bourque, 1993). Since the MNCs do not display regulatory volume changes in response to osmotic swelling or shrinkage (Zhang & Bourque, 2003), increases in osmolality lead to a sustained depolarization of the MNC membrane, an increased likelihood of cell firing in response to excitatory postsynaptic potentials, and an increase in hormone release. Initiation of phasic firing requires a depolarizing afterpotential (DAP) that follows evoked action potentials and can summate into the plateau potentials required for phasic firing in MNCs (Andrew & Dudek, 1983). The DAP may result from Ca2+-dependent activation of a non-selective cation current (Ghamari-Langroudi & Bourque, 1998, 2002) and/or the Ca2+-dependent inactivation of a K+ current (Li & Hatton, 1997). The MNCs also express hyperpolarizing afterpotentials, which are mediated by K+ currents (Bourque & Brown, 1987; Greffrath et al. 1998; Ghamari-Langroudi & Bourque, 2004) and could contribute to the cessation of firing required for burst termination. Termination of bursts may also involve a progressive decrease in the amplitude of the DAP and plateau potential caused by the auto-inhibitory actions of dynorphin released from MNC somata during bursts (Brown & Bourque, 2004).
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The acceleration of MNC firing that occurs as osmolality increases, and the transition that occurs between rapid continuous and phasic firing (Bourque & Renaud, 1984), may involve modulation of ion channels other than the SIC. We have therefore sought to determine whether other currents expressed in the MNCs are sensitive to changes in osmolality. We report here a slowly activating voltage gated cation current that increases in amplitude in response to increases in external osmolality. The properties of this current suggest that it could contribute to an afterpotential that regulates MNC firing patterns. This current might therefore play an important role in the regulation of VP and OT release. Some of these results have been presented in abstract form (Liu & Fisher, 2003).
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Methods
Animals and cell preparation
Male Long-Evans rats (200–300 g) were anaesthetized with halothane and killed by decapitation according to a protocol approved by the University of Saskatchewan Animal Care Committee. MNCs were isolated as previously described (Oliet & Bourque, 1992; Fisher & Bourque, 1995). The brain was removed and blocks of tissue containing most of the two supraoptic nuclei were obtained. The tissue blocks were incubated for 90 min at 34°C with an oxygenated (100% O2) Pipes solution (pH 7.1) composed of (mM): NaCl, 120; KCl, 5; MgCl2, 1; CaCl2, 1; Pipes, 20; glucose, 25; and containing trypsin (Type XI, 0.6 mg ml–1). The tissues were then transferred into oxygenated Pipes solution without trypsin for 30–120 min at room temperature. The tissues were gently triturated with fire-polished pipettes to disperse the cells onto glass-bottomed recording dishes for use the same day.
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Electrophysiological recording
Whole-cell patch-clamp recordings were made using fire-polished micropipettes (A-M Systems Inc., Carlsborg, WA, USA) prepared using a P-97 horizontal pipette puller (Sutter Instrument Co., Novato, CA, USA) and polished using a microforge (Narishige, Tokyo, Japan). Currents were recorded at room temperature using an EPC-9 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) operated with Pulse software (HEKA), and were low pass filtered (2 kHz). TEA/Na+ external solution (pH 7.35) was composed of (mM): NaCl, 120; CaCl2, 2; glucose, 10; Hepes, 10; TTX, 0.0002; TEA, 20; and 4-AP, 4 (Fisher & Bourque, 1995). The recording electrodes (2.5–5 M) were filled with a solution (pH 7.2) containing (mM): Tris, 110; MgCl2, 1; EGTA, 1.6; TEA, 40; Na2ATP, 2; and phosphocreatine, 14. In some experiments, external Na+ was replaced with N-methyl-D-glucamine (NMDG, 90 mM). The osmolality of the solutions were measured using a Vapro pressure osmometer (Wescor Inc., Logan, UT, USA) and adjusted to the desired osmolality by adding mannitol as required. All chemicals were from Sigma (St Louis, MO, USA).
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Data analysis
Tail current amplitudes were measured by subtracting the amplitude after the current was fully depolarized from that 20 ms after the end of the depolarization pulse. The current amplitude was divided by the cell capacitance to normalize for cell size in most experiments. Cell capacitance was measured automatically by the c-slow compensation function of the Pulse software. The mean capacitance of the responding MNCs was 12.5 ± 0.9 pF, which is close to that previously observed for a selected group of MNCs (15.2 ± 0.4 pF; Oliet & Bourque, 1992). Data are shown as the mean ± S.E.M. Statistical significance was estimated by Student's paired or unpaired t test as described. A P-value of less than 0.05 was regarded as significant.
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Results
Isolation of the osmosensitive cation current (OC)
When acutely isolated MNCs were patch clamped in the whole-cell mode using TEA/Na+ external solution, depolarizing steps were followed by a long lasting tail current (Fig. 1A). These solutions contained TTX to block Na+ currents, and K+ currents were diminished by the presence of 4-AP in the external solution, TEA in internal and external solutions, and by the absence of internal and external K+. Since ICa in MNCs has been shown to be fully deactivated within a few milliseconds (Foehring & Armstrong, 1996; Harayama et al. 1998), this tail current (which deactivated over about 400 ms) appeared to result from a current other than Ca2+ current. Depolarization from a holding potential of –80 mV to 0 mV for 400 ms resulted in a tail current with an amplitude greater than 20 pA (and as high as 400 pA) in 145 out of 175 MNCs. The amplitude of this tail current was sensitive to changes in the osmolality of the external solution (Fig. 1A and B). When the external osmolality was increased from 295 to 325 mosmol kg–1, a large increase in the amplitude of the inward tail current was frequently observed, as well as a small increase in inward current during the depolarizing step (Fig. 1A, left). Overall, about 60% of the cells (79 out of 134 cells) underwent a marked increase in tail current amplitude; the mean tail current density in these sensitive cells doubled from 5.2 ± 0.9 pA pF–1 in 295 mosmol kg–1 to 10.5 ± 1.4 pA pF–1 in 325 mosmol kg–1 (P < 0.001; Fig. 1A, right). Curve fitting of the tail current in 325 mosmol kg–1 showed that the current deactivated with a time constant of 75.0 ± 9.1 ms. The change in amplitude was reversible when the external solution was returned to 295 mosmol kg–1 (data not shown). Furthermore, the amplitude of this tail current decreased when the osmolality of the external solution was decreased from normal (295 mosmol kg–1) to hypotonic (265 mosmol kg–1; Fig. 1B). Figure 1B (left) shows the amplitudes of currents evoked in a single MNC in 295, 265 and 325 mosmol kg–1; Fig. 1B (right) shows the time course of these changes in the same cell. Similar results were seen in 8 out of 15 cells tested. Since the osmolality was changed using a gravity-fed perfusion system, it is likely that the rate of change in current amplitude was determined largely by the perfusion rate. We will henceforth refer to the current whose amplitude is changed with changes in external osmolality as the osmosensitive current or OC. The current required to hold the cells at –80 mV was unchanged by changes in the external osmolality, which suggests that the SIC is blocked under these conditions (probably by the presence of internal TEA; authors' unpublished observations).
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A, left, currents evoked by depolarizing an MNC from –80 mV to 0 mV in a 295 mosmol kg–1 solution and then in a 325 mosmol kg–1 solution. Note the large increase in the amplitude of the tail current. Right, the mean tail current density in 79 responding cells before and after increasing the osmolality of the external solution from 295 to 325 mosmol kg–1 (P < 0.001). B, left, currents evoked by depolarizing steps in an MNC in 295, 265 and 325 mosmol kg–1 solutions. Right, the time course of changes in tail current amplitude (normalized to the maximum evoked amplitude) during the noted changes in external osmolality.
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We tested the ionic basis of the OC by replacing external Na+ with NMDG. Figure 2A shows that the OC density in 325 mosmol kg–1 TEA/Na+ solution (12.5 ± 3.676 pA pF–1) was nearly abolished after Na+ was replaced with NMDG (0.522 ± 0.481 pA pF–1; n = 6). There was also a small decrease in inward current during the depolarizing step. These data support the conclusion that the OC is a cation current that increases in amplitude as the external osmolality is increased, and that Na+ is the major charge carrier under these conditions. In support of this conclusion, including 40 mM Na+ in the internal solution (by replacing 40 mM Tris) caused a significant decrease in the amplitude of the tail current (to 6.8 ± 1.0 pA pF–1 at –80 mV, n = 8, compared to 10.5 ± 1.4 pA pF–1 as shown in Fig. 1), presumably because the driving force for Na+ was decreased.
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A, left, currents evoked by depolarizing an MNC from –80 mV to 0 mV in a 325 mosmol kg–1 TEA/Na+ solution and after replacing Na+ with NMDG. Note that the tail current amplitude is dramatically decreased and that there is a small decrease in the inward current evoked during the step. Right, the mean tail current density in 6 cells before and after replacing Na+ with NMDG (P < 0.001). B, left, currents evoked by depolarizing an MNC from –80 mV to 0 mV in 295 mosmol kg–1 and 325 mosmol kg–1 TEA/NMDG solutions. There was no change in the amplitude of evoked Ca2+ current or tail current. Right, the mean Ca2+ current density in 5 cells in 295 and 325 mosmol kg–1 TEA/NMDG solutions. C, left, currents evoked in 295 and 325 mosmol kg–1 solutions with 5 mM BAPTA included in the internal solution. Right, tail current density in 5 cells in 295 and 325 mosmol kg–1 solutions with 5 mM BAPTA included in the patch pipette.
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To test whether Ca2+ currents are sensitive to changes in osmolality, Ca2+ currents were evoked in TEA/NMDG external solution. Under these conditions, there was no change in amplitude of either the Ca2+ current or the tail currents that followed (Fig. 2B). The Ca2+ currents in MNCs therefore do not appear to be osmosensitive.
To test whether the OC is activated by an increase in intracellular Ca2+, we activated the OC in the presence of BAPTA in the pipette solution. BAPTA caused no discernable difference in the amplitude of the OC or its increase in amplitude in response to increased osmolality (Fig. 2C). These data suggest that the OC is not dependent on increases in intracellular Ca2+ for activation and that it is primarily voltage dependent.
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Kinetics and Ca2+ dependence of the OC
Figure 3A shows that the amplitude of the tail current is time dependent and increases as the length of depolarizing steps increases over a range from 10 to 500 ms. The traces above show currents evoked by steps from –80 mV to 0 mV of the indicated lengths and the plot shows the mean results obtained from 15 cells. The tail current was fully activated by depolarizing pulses of about 400 ms (Fig. 3A). The OC therefore activates and deactivates over hundreds of milliseconds.
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A, tail current density following depolarization of different lengths. Cells in 325 mosmol kg–1 TEA/Na+ solution were stepped from a holding potential of –80 mV to 0 mV for different lengths of time (from 10 ms to 500 ms) and then returned to –80 mV. The traces above show the currents evoked by these steps and the plot below shows the mean results from 15 cells. B, tail current density evoked by steps to different voltages. Cells were stepped from a holding potential of –80 mV to a series of potentials between –70 mV and +20 mV for 400 ms (in 10 mV increments every 1.5 s) and then returned to –80 mV in 295 and then 325 mosmol kg–1 TEA/Na+ solution. The traces above show families of tail currents (commencing 20 ms after the depolarizing step) evoked in the same cell in 295 and 325 mosmol kg–1 solutions. The plot below shows the mean current–voltage relationship for 9 cells. The increase in current caused by the change to 325 mosmol kg–1 solution is plotted using triangles.
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Figure 3B plots the mean current–voltage relationships for activation of the tail current in 295 and 325 mosmol kg–1 solutions in nine MNCs. The OC was activated by steps to potentials greater than –60 mV and fully activated at 0 or +10 mV. These data support the conclusion that OC activation is primarily dependent on voltage, rather than Ca2+ influx, since the OC is activated at potentials at which there is no measurable Ca2+ current under these conditions, and does not appear to decrease in amplitude at potentials at which the Ca2+ current has decreased in amplitude (Ca2+ current peaks at –10 mV in MNCs and declines at higher potentials (Fisher & Bourque, 1995). Furthermore, OC amplitude does not correlate well with the amplitude of Ca2+ current in individual MNCs, and is stable over time despite the rundown of Ca2+ current (data not shown).
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Pharmacological properties
The amplitude of the OC was reversibly inhibited by the addition of 1 mM Cs+ in 325 mosmol kg–1 TEA/Na+ external solution (Fig. 4; n = 18). The traces in Fig. 4A (left) show the tail current before, during, and after the addition of Cs+. Ca2+ currents did not appear to be affected. Figure 4A (right) shows the tail current density before (7.572 ± 5.957 pA pF–1) and after (0.882 ± 0.7015 pA pF–1) addition of Cs+, indicating an 88% decrease in amplitude. When Cs+ (10 mM) was included in the internal solution, the tail current in amplitude was very low in 295 mosmol kg–1 solution (1.48 ± 0.35 pA pF–1, P > 0.05) and was not significantly increased by perfusion with 325 mosmol kg–1 solution (1.79 ± 0.31 pA pF–1) in four cells (Fig. 4B). The OC is therefore sensitive to both external and internal Cs+. The inclusion of Cs+ in internal solutions used in previous studies of voltage gated Ca2+ currents in MNCs (Foehring & Armstrong, 1996; Harayama et al. 1998) may help to explain why this tail current was not observed.
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A, left, currents evoked by depolarizing an MNC from –80 mV to 0 mV in 325 mosmol kg–1 solutions before, during and after addition of 1 mM Cs+. Right, the mean tail current density of 18 cells before and after addition of 1 mM Cs+ (P < 0.001). B, left, currents evoked in 295 and 325 mosmol kg–1 solutions with 10 mM Cs+ included in the internal solution. Right, the mean tail current density of 4 cells in 295 and 325 mosmol kg–1 solutions with 10 mM Cs+ included in the internal solution (P > 0.05). C, left, currents evoked in 325 mosmol kg–1 external solution before, during and after addition of 0.25 mM Ba2+. Right, the mean tail current density of 9 cells before and after addition of 0.25 mM Ba2+ (P < 0.001). D, left, currents evoked in an MNC with an internal solution containing 5 mM K+ in 295, 325 and 325 mosmol kg–1 external solution in which 40 mM Na+ was replaced with 40 mM K+. Right, the mean tail current densities in MNCs recorded with an internal solution containing 5 mM K+ in 295, 325 and 325 mosmol kg–1 external solution containing 40 mM K+ (n = 11). The addition of 40 mM K+ caused a significant increase in tail current density (P < 0.001).
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The traces in Fig. 4C (left), show the currents evoked before, during, and after the addition of Ba2+ in 325 mosmol kg–1 external solution. Figure 4C (right) shows the mean tail current density before and after application of Ba2+. The OC amplitude was reversibly blocked from 7.81 ± 1.69 pA pF–1 to 0.996 ± 0.104 pA pF–1 by 250 μM Ba2+ in nine MNCs (Fig. 4C, right). This represents an 89% blockade. These results indicate that the OC is sensitive to external Ba2+.
, 百拇医药 The block of the OC by Ba2+ and Cs+ suggests the possibility that the OC could be a K+ current, whose ion selectivity is altered in the absence of K+ such that Na+ becomes permeant. In support of this hypothesis is the observation (Fig. 4D) that the addition of a small concentration of K+ (5 mM) to the internal solution results in a decreased tail current density in 295 mosmol kg–1 solution (0.73 ± 0.27 pA pF–1) and prevents the increase in current when the solution is changed to 325 mosmol kg–1 (0.86 ± 0.26 pA pF–1; n = 11). Furthermore, addition of 40 mM K+ to the 325 mosmol kg–1 external solution in exchange for 40 mM Na+ leads to the appearance of an inward tail current (5.24 ± 0.45 pA pF–1; n = 11). These data support the hypothesis that the OC is a K+ current.
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Discussion
The identification in the somata of MNCs of an osmosensitive, voltage gated, slowly activating cation current is a novel finding that could have important implications in the regulation of MNC firing and neuropeptide release. The block of the OC by Cs+ and Ba2+, and the decrease of Na+ permeability by the inclusion of K+ in the internal solution suggest that the OC is most likely to be a K+ current. Our data suggest that the OC is clearly different from the SIC, which is a mechanosensitive non-selective cation channel that is relatively insensitive to voltage (Oliet & Bourque, 1993) and which is not sensitive to Cs+ (C. W. Bourque, personal communication). The OC is also clearly distinct from a stretch-sensitive voltage-insensitive K+ leak channel that has been identified using single channel cell-attached recordings of MNC somata (Han et al. 2003). The OC, in contrast to both of these currents, appears to be a voltage gated channel whose amplitude is modulated by changes in the external osmolality. It remains to be seen whether this effect is mediated by a direct action of membrane tension on the channel, or is mediated via a second messenger system that is itself osmosensitive.
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Our observation that only about 60% of the MNCs expressed currents that responded to changes in osmolality is unexplained. Since VP and OT MNCs respond differently to osmotic stimulation, it is possible that a greater proportion of one type expresses the OC. Whole-cell patch- clamp studies of MNCs in slices have demonstrated, however, that MNCs can be converted from phasic to continuous firing and vice versa by altering internal Ca2+ buffering (Li et al. 1995). These data suggest that all MNCs express the currents necessary to support phasic firing and that differences in firing patterns between OT and VP MNCs result in part from differences in the levels of Ca2+ binding proteins. The distribution of the OC in VP and OT MNCs will therefore need to be determined.
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Although the physiological role of the OC is not yet clear, it seems reasonable to speculate that it is involved in mediating the changes in MNC firing that occur in response to changes in external osmolality. If the OC is a Na+ current or a non-selective cation current, its activation could contribute to an increase in the size of the DAP and/or the plateau potential during hyperosmolality. Increases in osmolality, however, do not appear to change the amplitude of the DAP (Bourque, 1989). If the OC is a K+ current, it might contribute to the afterhyperpolarizations (AHPs) observed in MNCs. These include a medium AHP (mAHP) lasting 200–500 ms (Bourque & Brown, 1987; Armstrong et al. 1994; Kirkpatrick & Bourque, 1996), and a slow AHP (sAHP), which decays over seconds (Greffrath et al. 1998). Both of these currents are important in regulating firing patterns. Blockade of the mAHP with apamin causes a decrease in burst length (Kirkpatrick & Bourque, 1996) and inhibition of the sAHP by muscarinic agonists increases burst length (Ghamari-Langroudi & Bourque, 2004). Both the mAHP and the sAHP are selectively elevated in OT MNCs during lactation (Teruyama & Armstrong, 2005), suggesting that an increase in AHP amplitude may be an adaptation that favours burst firing. Although both these currents are largely Ca2+ dependent, it is possible that the OC could contribute to the mAHP and/or the sAHP, and that an increase in its amplitude during increases in external osmolality might influence firing patterns. Future studies will pursue elucidation of the ionic selectivity and molecular nature of the OC, the molecular mechanism underlying its osmosensitivity, and its physiological role.
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