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Taurine and the control of basal hormone release from rat neurohypophysis
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Experimental Neurology 183 (2003) 330 –337
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Taurine and the control of basal hormone release from rat neurohypophysis Zhilin Song and Glenn I. Hatton* Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA Received 27 November 2001; revised 9 January 2003; accepted 16 January 2003
Abstract Pituicytes of pituitary neural lobe are rich in the amino acid taurine, which they release upon hypoosmotic stimulation. As a generally inhibitory amino acid, taurine is thought to activate receptors on neural lobe nerve terminals and exert some control over hormone release. Previous work has shown the presence of glycine and GABAA receptors in neural lobe, both of which have affinity for taurine. Using a perifused explant system, we studied the effects of taurine activation of glycine and GABAA receptors on basal hormone release. Somewhat surprisingly, taurine induced increases in basal release of both vasopressin and oxytocin. Taurine-induced increases in oxytocin release were blocked by bicuculline, suggesting involvement of GABAA receptors. Increases in vasopressin release were not blocked by bicuculline, indicating involvement of receptors other than GABAA. Although combined bicuculline and strychnine, an antagonist at most glycine receptors, also did not block increased vasopressin release, picrotoxin (a Cl⫺ channel blocker) was effective in blocking increases in both vasopressin and oxytocin release. The other receptor(s) involved in taurine actions is postulated to be strychnine-insensitive glycine receptors. Thus, taurine in neural lobe may act via both a GABAA receptor and one or more types of glycine receptors to depolarize nerve terminal membranes under basal conditions. Taurine-induced partial depolarization resulting in Na⫹ channel inactivation is probably responsible for its previously observed inhibition of stimulated hormone release from neural lobe. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Pituitary neural lobe; Pituicytes; Magnocellular hypothalamo-neurohypophysial system; Vasopressin; Oxytocin; GABAA receptor; Glycine receptor
Introduction Taurine, a GABA-like amino acid, is generally regarded as an osmoregulator of the cell. Taurine is present in supraoptic nucleus (SON) of the magnocellular hypothalamoneurohypophysial system (mHNS). In SON, taurine was found predominantly in astrocytes, with traces in occasional dendrites and somata, suggesting uptake by these latter cellular compartments (Decavel and Hatton, 1995). Astrocytes have been shown to express the synthetic enzyme for taurine, cysteine sulfinic acid decarboxylase, and to be capable of rapid taurine synthesis (Almarghini et al., 1991; Beetsch and Olson, 1998). Ultrastructural studies revealed that taurine-rich astrocytes completely engulfed many of the * Corresponding author. Fax: ⫹1-909-787-2967. E-mail address: [email protected] (G.I. Hatton).
axodendritic synaptic contacts in the SON dendritic zone (Decavel and Hatton, 1995), and that astrocytic processes covering the somata frequently contained large amounts of taurine. This suggests that taurine released from astrocytes into the space between the glial and neural elements could attain rather high concentrations at the neural membrane. It was demonstrated that taurine was released from SON glial cells under basal conditions (normo- and hypoosmotic), indicating that taurine may have a tonic inhibitory influence on SON firing, particularly of vasopressin (VP) cells (Deleuze et al., 1998). This strongly suggests that astrocytederived taurine acts as a modulator in the control of hormone release. The involvement of glycine receptors (glyRs) was suggested by the much higher affinity of glyRs for taurine compared with that of GABAA receptors (GABAARs) for taurine in the SON (Hussy et al., 1997; Randle and Renaud, 1987). Physiological studies revealed
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that taurine released from SON glia acts on glyRs to induce neuronal hyperpolarization via increased Cl⫺ conductance, while locally applied strychnine, a selective glyR antagonist, increased electrical activity of VP neurons in normally hydrated rats. This increase was greatly enhanced in waterloaded rats (Hussy et al., 1997). Together, these studies suggest that taurine is tonically released by astrocytes in the SON under normo- to hypoosmotic conditions, and that the amount of taurine released is sufficient to play a role in the overall inhibition of SON neurons. Glial withdrawal during mHNS activation, then, is tantamount to removal of one source of tonic inhibition induced by taurine via glyRs at the soma-dendritic level. Interestingly, in neural lobe (NL) of the mHNS, where axons and terminals originating from SON reside and hormones are released, taurine was found to be rich and exclusively present in pituicytes. Furthermore, taurine was released from NLs upon hypoosmotic stimulation (Miyata et al., 1997). It is reasonable to postulate that taurine may also act directly on the NL axons/terminals to regulate hormone release as it does in the SON. Since the NL is the final site in mHNS at which control of hormone release into the blood can take place, it is important to understand the mechanisms operating at this level. While it is reasonable to suppose that some of the same receptors would be expressed on the terminals as are expressed at the soma-dendritic level of these neurons, it is also possible that they are different. Central GABAergic innervation of NL has been revealed by biochemical and ultrastructural immunocytochemical studies (Buijs et al., 1987; Oertel et al., 1982). Existence of GABAARs on nerve terminals in NL was demonstrated electrophysiologically and blockade of action potential propagation or reduction in amplitude of compound action potentials was achieved by GABAAR activation (Jackson and Zhang, 1995; Zhang and Jackson, 1993, 1995a, 1995b; Zingg et al., 1979). Little is yet known about glyRs in NL. Since taurine at high concentrations can activate GABAARs, taurine’s action on neural membrane in NL could be mediated via either one or both of these two receptors. In the present study, we demonstrated an enhancing effect on basal hormone release as a result of activating GABAARs by GABA and glyRs by glycine. Taurine had effects similar to those of GABA on basal VP and oxytocin (OT) release in NL and its effects were blocked by picrotoxin, a Cl⫺ channel blocker. It is likely that the taurine effects on OT release in NL were mediated via GABAARs, whereas its effects on VP release were mediated via both GABAARs and glyRs.
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the Institutional Animal Care and Use Committee at the University of California, Riverside. Superfusion of NL explants Brains were removed from rats and pituitaries were taken out. Anterior lobe was removed. As much intermediate lobe as possible was removed without damaging the NL. NL explants were perifused individually in closed culture chambers (0.5 mL) at 37°C with oxygenated (95% O2, 5% CO2) ACSF at a rate of 3.75 ml/h. The ACSF consists of the following components (in mM): NaCl 126, NaHPO4 1.3, NaHCO3 26, KCl 5, CaCl2 2.4, MgSO4 1.3, glucose 10, and 3[N-morpholino] propane sulfonic acid buffer 5, pH 7.4. This organic buffer, in combination with the bicarbonate buffer, has been found in our earlier studies to provide better pH stability in long duration experiments than use of the bicarbonate buffer alone. The osmolality of this ACSF is 310 mOsm/l, with an effective osmolality of 300 mOsm/l. To better preserve and prolong tissue viability, ACSF was modified with 0.2 mM ascorbic acid and 0.2 mM thiourea (Stern and Armstrong, 1995). Bacitracin (400 mg/l) was also added to the ACSF to prevent hormone degradation (Sladek and Armstrong, 1987). After 3 h of equilibration, explants were maintained either continuously under ACSF as control or ACSF fortified with different reagents. Outflow was collected individually at 15-min intervals using a fraction collector maintained at 4°C. Data analysis Both VP and OT contents in the perifusate were determined by a sensitive enzyme immunoassay (EIA) kit (Assay Designs, Inc., Ann Arbor, MI). Basal hormone release was calculated for each explant as the mean hormone release during the period just before drug exposure. VP and OT release were expressed as percentage of the basal release for that explant. The results were expressed as mean ⫾ SEM. Analysis of variance (ANOVA) with repeated measures was performed using Sigma Stat in order to evaluate differences in VP and OT release as a result of drug treatment. Subsequent ANOVA and post hoc Student Neuman–Keuls tests were performed to establish specific group differences at individual time points.
Results Taurine effects on VP and OT release
Materials and methods Animals Male Sprague–Dawley rats 45– 60 days old were used in all experiments, and were killed by decapitation. All experimental protocols were done within National Institute of Health guidelines for animal research and were approved by
We evaluated the direct functional impact of taurine on basal hormone release. To determine an optimal concentration, taurine at 1, 10, or 50 mM was administered sequentially to NL explants for 0.5 h each. Since a consistent, near maximal response was achieved at 10 mM (data not shown), this concentration was deemed appropriate and was adopted for subsequent experiments involving taurine. In this and
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subsequent experiments, both VP and OT release were monitored from the same NLs. Taurine significantly increased VP release throughout the treatment period (F ⫽ 26.219, P ⬍ 0.001) (Fig. 1A). Basal VP release for taurine-treated and control groups was 31.8 ⫾ 6.6 and 27.4 ⫾ 4.9 pg/mL, respectively. OT release was also significantly increased by taurine treatment (F ⫽ 22.672, P ⬍ 0.001) (Fig. 1B). Basal OT release for taurine-treated and control groups was 46.3 ⫾ 7.4 and 75.2 ⫾ 17.3 pg/ml, respectively. Glycine effect on VP release Glycine’s effect on basal hormone release was evaluated. After a 3-h equilibration period, NL explants were exposed to either continued control ACSF or to ACSF containing Fig. 2. VP release upon glycine treatment. Glycine at 300 M was applied in one group (Œ). Release results were compared with that of a group that was maintained in control ACSF (F). Glycine treatment significantly increased VP release from neural lobe compared with control group. *P ⬍ 0.05 for mean comparison at individual time points between these two groups. Arrows indicate when glycine actually reached the explants.
300 M glycine. A significant increase in VP release was observed with glycine treatment compared to that of control explants (F ⫽ 36.842, P ⬍ 0.001) (Fig. 2). Basal VP release for glycine-treated and control groups was 17.0 ⫾ 2.2 and 22.5 ⫾ 3.9 pg/ml, respectively. OT release was not monitored in this experiment, since activation of glyR seems to have no apparent effect on basal OT release from related experiments (data not shown). GABA effects on VP and OT release Next we assessed the effect of GABAR activation by GABA on basal hormone release from the NL. Explants were equilibrated for 3 h and then perifused continuously with control ACSF or exposed to ACSF containing 100 M or 1 mM GABA sequentially for 0.5 h each. Results are shown in Fig. 3. As seen in Fig. 3A, GABA treatment significantly increased VP release (F ⫽ 11.490, P ⫽ 0.007). Basal VP release for GABA-treated and control groups was 40.2 ⫾ 6.8 and 45.9 ⫾ 12.8 pg/ml, respectively. Exposure to 1 mM GABA also produced a significant increase in OT release (F ⫽ 10.517, P ⫽ 0.008) (Fig. 3B). Basal OT release for GABA-treated and control groups was 56.5 ⫾ 6.8 and 82.6 ⫾ 7.4 pg/ml, respectively. It appears that GABAR activation in NL can increase basal release of both VP and OT, with the VP response being more sensitive and robust than the OT response. Fig. 1. VP and OT release upon taurine treatment. Taurine at 10 mM was applied in one group (F). Release results were compared with that of control group that was maintained in control ACSF (Œ). Both VP and OT release were monitored. Both VP (A) and OT (B) basal releases were significantly increased by taurine treatment during the entire experiment period (1.5 h). *P ⬍ 0.05 (Student Newman–Keuls test) for mean comparison at individual time points in both (A) and (B). Arrows indicate when taurine actually reached the explants.
Bicuculline and strychnine effects on taurine-induced hormone release Whether the taurine effect on basal hormone release is via GABAAR or glyR activation is unknown. To elucidate this, explants were treated with 10 mM taurine, then taurine
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increase in OT release was due to taurine treatment alone. Together with BIC or a combination of BIC and strychnine, taurine failed to change OT release significantly. Basal OT release for drug-treated and control group was 99.5 ⫾ 11.3 and 106.0 ⫾ 6.1 pg/ml, respectively. Both the VP and the OT responses to taurine treatment showed a slightly longer time lag in this experiment compared to the previous one (cf. Figs. 1 and 4). This was probably due to relatively larger basal VP and OT release in this experiment. Picrotoxin effect on taurine-induced hormone release Although combined BIC and strychnine were not able to block taurine-induced increase in basal VP release
Fig. 3. VP and OT release upon GABA treatment. GABA at 100 M and 1 mM were applied sequentially for 0.5 h each in one group (F). Release results were compared with that of control group that was maintained in continuous ACSF (Œ). (A) GABA at 1 mM significantly increased VP release at both time points (*P ⬍ 0.01 by Student Newman–Keuls test). GABA at 100 M had a tendency to increase VP release. P values for individual time points are as follows: 5, 0.226; 6, 0.097; 7, 0.002; 8, ⬍0.001. (B) GABA at 1 mM increased OT hormone release, but this increase was statistically significant only at the last time point (* P ⬍ 0.05 by Student Newman–Keuls test).
plus 50 M bicuculline (BIC, a GABAAR antagonist), followed by taurine plus a combination of BIC and 10 M strychnine (a glyR antagonist), 0.5 h each. Results are shown in Fig. 4. VP release was elevated by taurine treatment and this effect was not abolished by coapplication of BIC or BIC/strychnine along with taurine (F ⫽ 17.520, P ⫽ 0.002) (Fig. 4A). Basal VP release for drug-treated and control groups was 40.3 ⫾ 5.7 and 36.2 ⫾ 3.6 pg/ml, respectively. For OT release, ANOVA revealed a statistically significant interaction between group and time (F ⫽ 3.487, P ⫽ 0.003) (Fig. 4B). Post hoc Student Newman– Keuls test determined the significance levels for mean comparison at individual time points as follows: 4, P ⬍ 0.001; 5, P ⬍ 0.087; 6, P ⬍ 0.297; 7, P ⬍ 0.605; 8, P ⬍ 0.132. The
Fig. 4. Effect of bicuculline and strychnine on taurine-induced VP and OT release. In one group (F), taurine 10 mM alone was applied first, followed by taurine plus 50 M bicuculline (BIC) and then taurine together with BIC and 10 M strychnine, 0.5 h each. Release results were compared with that of control group that was maintained in control ACSF (Œ). (A) Taurine increased VP hormone release and this effect was not blocked by either BIC alone or BIC plus strychnine (*P ⬍ 0.05 by Student Newman–Keuls test). (B) Taurine at 10 mM significantly increased OT release (*P ⬍ 0.001 by Student Newman–Keuls test). This effect was blocked by BIC alone or BIC plus strychnine.
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rine (probably via both GABA A Rs and strychnineinsensitive glyRs), influence hormone release in NL. In Figs. 5A (VP) and B (OT), ANOVA results for all three groups were as follows: for VP, F ⫽ 0.000247, P ⫽ 1.000; for OT, F ⫽ 0.352, P ⫽ 0.710. Basal VP release (A) for PIC, PIC ⫹ taurine and control groups was 29.0 ⫾ 10.0, 33.2 ⫾ 4.5, and 37.5 ⫾ 4.1 pg/ml, respectively. Basal OT release (B) for PIC, PIC ⫹ taurine and control groups was 42.9 ⫾ 13.7, 59.2 ⫾ 10.6, and 70.2 ⫾ 13.6 pg/ml, respectively. Taurine effect on VP and OT release in Ca2⫹-free perifusion ACSF
Fig. 5. Picrotoxin effect on taurine-induced hormone release. Picrotoxin (PIC 100 M) alone (Œ) or PIC together with 10 mM taurine (■) was included in perifusion medium. Release results from these two groups were compared with that of control group (F) that was maintained continuously in ACSF. PIC alone did not change either VP (A) or OT (B) basal hormone release. In either case (VP or OT), the expected increase in hormone release upon 10 mM taurine treatment was not seen when PIC 100 M was present. Arrows indicate when the drugs actually reached the explants.
(Fig. 4A), it is still possible that taurine effect on VP release was mediated via a combination of glyR and GABAAR, since a strychnine-insensitive population of glyRs might exist in NL (Kushe et al., 1990; Song et al., 2001). To investigate this possibility, picrotoxin (PIC), a specific chloride channel blocker, known to block both GABAAR- and glyR-operated chloride channels, was tested for its ability to block taurine-induced hormone release. PIC (100 M) alone did not change basal release of either hormone (Fig. 5). As predicted, increase in neither VP nor OT release in response to 10 mM taurine treatment was seen when 100 M PIC was present. This result suggests that chloride channels, activated by tau-
To demonstrate Ca2⫹-dependence of taurine effects on release, VP and OT release upon taurine treatment was evaluated in Ca2⫹-free perifusion ACSF. The 2.4 mM CaCl2 · 2H2O in regular ACSF was replaced by 2.4 mM MgCl2 · 6H2O to maintain osmolar and Cl⫺ balance. The explants were equilibrated for 3 h as usual. This procedure also flushed out endogenous extracellular free Ca2⫹. Then taurine at 10 mM was added to the perifusion medium of one group, while control group was continuously maintained in control ACSF. Results are shown in Fig. 6. Taurine failed to induce any increase in either VP (Fig. 6A) or OT (Fig. 6B) hormone release, indicating that extracellular Ca2⫹ is required for taurine to stimulate either VP or OT basal hormone release. ANOVA results were as follows: for VP, F ⫽ 0.203, P ⫽ 0.663; for OT, F ⫽ 1.860, P ⫽ 0.695. Basal VP release (Fig. 6A) for taurine and control groups was 9.0 ⫾ 1.9 and 11.0 ⫾ 2.7 pg/ml, respectively. Basal OT release (Fig. 6B) for taurine and control groups was 113.7 ⫾ 16.5 and 115.1 ⫾ 17.5 pg/ml, respectively.
Discussion Data from the present studies indicate that taurine in the NL increases basal release of both VP and OT. Since taurine is an agonist for both GABAARs and glyRs, it was postulated that taurine could act through GABAARs and/or glyRs to exert its control on hormone release at nerve terminals of the NL. Moreover, we demonstrated the effect of activation of GABAARs or glyRs on basal hormone release. Further studies with receptor antagonists were conducted to elucidate the underlying mechanisms. Role of GABAAR in VP and OT release in NL GABAARs have long been implicated as having a functional role in regulation of hormone release from the NL. The underlying mechanisms involved, however, are not clear, and results from previous studies on hormone release
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Fig. 6. VP and OT release upon taurine treatment in Ca2⫹-free ACSF. Taurine at 10 mM was applied in one group (F). Release results were compared with that of control group that was maintained in control ACSF (Œ). Both VP and OT release were monitored. In either case (VP (A) or OT (B)), the expected increase in hormone release upon 10 mM taurine treatment was not seen when ACSF was free of Ca2⫹. Arrows indicate when taurine actually reached the explants.
in NL were controversial. GABAAR activation by GABA or other agonists like muscimol inhibited intense electrical or excess K⫹ stimulation-induced VP and OT release (Dyball and Shaw, 1978; Magnusson and Meyerson, 1993; Saridaki et al., 1989). Conversely, VP/OT release induced by submaximal electrical stimulation was greatly enhanced by isogauvacin, a GABAAR agonist (Fjalland et al., 1987). However, GABA did not affect spontaneous hormone release in previous studies (Dyball and Shaw, 1978; Iversen et al., 1980). In our current studies, an increase in both VP and OT basal hormone release was observed with GABA treatment of the NL, confirming the opposite effects of GABAAR activation on hormone release in NL under strong stimulation vs basal conditions. Similarly, GABA inhibited stimulated melanocyte stimulating hormone (MSH) release from intermediate lobe (IL), but increased nonstimulated
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MSH release (Schlichter et al., 1984; Tomiko et al., 1983). How does this bipolar effect of GABAAR activation on hormone release occur? One answer comes from electrophysiological analyses in NL terminals. Patch– clamp studies revealed a relatively high intracellular Cl⫺ concentration (⬃20 mM) in nerve terminals of NL, which resulted in a ⫹14-mV membrane depolarization when activated by 50 M GABA (Zhang and Jackson, 1993, 1995a, 1995b). This amount of depolarization did not reach the threshold for Na⫹ channels to generate action potentials but instead inactivated Na⫹ channels, and thus blocked propagation of action potentials down to the nerve terminals (Jackson and Zhang, 1995). The blockade of action potential propagation may contribute to the inhibition of electrically stimulated hormone release by GABAR activation. On the other hand, this amount of depolarization by GABAR activation alone might be big enough to activate some types of voltagesensitive Ca2⫹ channels, leading to a rise of intracellular Ca2⫹ concentration in nerve endings that was responsible for increased basal hormone release. This explanation is supported by recent reports that a weakly depolarizing Cl⫺ current induced by activation of presynaptic GABAARs or glyRs was able to activate Ca2⫹ channels and enhance transmitter release (Jang et al., 2001a, 2001b; Turecek and Trussell, 2001). It is further evidenced by our observation that taurine effects on basal VP and OT release depended on extracellular Ca2⫹. This kind of action on GABAAR activation may also account for the increase of MSH release from IL under basal conditions. However, blockade of action potential propagation by a partial depolarization from GABAAR activation does not address how GABAR activation suppressed excess K⫹-induced hormone release in NL (Hussy et al., 2001). Electrophysiological studies on superior cervical sympathetic ganglion neurons revealed that GABA receptor activation resulted in a depolarization of neuronal membrane and a great reduction in membrane resistance. This action provides a “shunt” to the circuit across neuronal membrane and leads to a reduction in amplitude of orthodromic stimulation-induced spikes or direct stimulation-induced synaptic potentials (Adams and Brown, 1975; Brown and Marsh, 1978). In the adjacent IL, GABAR activation also caused a reduction in spike generation and reduced the amplitude of excess (100 mM) K⫹-induced membrane depolarization via a partial depolarization (Taraskevich and Douglas, 1982). This can account for an increase in MSH release under basal conditions but a suppression of excess K⫹-induced MSH release in IL by GABAR activation. Actions of this sort with GABAR activation may also explain why in NL, GABAAR activation increased basal or submaximal stimulation-induced, but reduced K⫹-stimulated, VP and OT release. Our observation of GABA-induced increased basal hormone release in NL is consistent with these findings and supplies further supporting evidence for the postulated mechanisms of GABAAR action on hormone release in NL.
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Taurine effect on VP and OT release in NL and the role of GABAAR Although it was reported previously that a lower concentration of taurine had no apparent effect on basal hormone release in NL (Hussy et al., 2001), in our current studies, a higher concentration of taurine administered exogenously increased both VP and OT hormone release under basal conditions, mimicking the effect of activating GABAARs by GABA itself. Furthermore, the increase in OT release by taurine was blocked by the GABAAR antagonist bicuculline. This strongly suggests that taurine in NL was capable of altering hormone release via activation of GABAARs. Its effect on hormone release should be similar to that of GABA, that is, to slightly increase basal hormone release, but suppress stimulated hormone release. Taking into account that taurine is abundant in pituicytes and it is released in response to a decrease in osmotic pressure (Miyata et al., 1997), it may be reasonable to postulate that in vivo, taurine could be released in NL in response to a reduction in osmotic pressure, and thus limit stimulated hormone release. What is its relation to system function? The mHNS can be aroused by many different stimuli, such as blood volume loss and emotional changes. These arousing signals are converted into action potentials in magnocellular neurons, which are transmitted to the nerve endings in NL. Depolarization of terminal membrane triggers hormone release in response to these stimuli. The resulting elevation in plasma VP levels could increase water retention by the kidneys and thus decrease systemic osmotic pressure. This decrease in osmotic pressure would lead to taurine release from pituicytes and its subsequent action on nerve terminals would block the further propagation of action potentials down to nerve endings. Taurine would therefore serve as a “shut off” switch for VP/OT hormone release. Involvement of glyRs in taurine regulation of VP/OT hormone release in NL Taurine action in the NL may be mainly mediated via GABAARs (as appears to be the case for OT), but a role for glyRs could not be excluded. First, the existence of glyRs in NL was demonstrated by immunocytochemical studies (Hussy et al., 2001; Song et al., 2000). Western blot studies also showed the existence of glyRs in NL (Song et al., 2001). Since taurine is an agonist at glyRs and the affinity of glyRs for taurine is even higher than that of GABAARs for taurine, it may be that glyRs are important taurine targets in the NL. Second, although the taurine-induced increase in basal OT release was blocked by BIC, that GABAAR antagonist did not block the increase in basal VP release in response to taurine. Hence, there must be at least one other type of receptor involved in regulation of VP release, and the glyR appears to be an ideal candidate. Third, glycine was able to increase basal VP release from NL in our studies. Taken together, this evidence suggests a role for
glyRs in taurine action on nerve terminals of NL. Direct evidence for the involvement of glyRs in taurine action in NL comes from a recent report by Hussy et al. (2001), in which taurine was able to inhibit high K⫹-induced VP release via a strychnine-sensitive glyR. All the evidence above indicates involvement of a strychnine-sensitive glyR in taurine actions, although glyR may have played a much lesser role in taurine regulation of OT release, as shown by our result that taurine-increased OT basal release was blocked by BIC and supported by the fact that much lower expression of glyR was reported in OT nerve endings (Hussy et al., 2001). Since combined treatment with BIC and strychnine did not block (or just partially blocked) taurine-induced increase in basal VP release, there has to be a third component other than GABAAR and strychninesensitive glyRs involved in taurine actions. In one of our experiments, the chloride channel blocker picrotoxin successfully blocked the expected taurine-induced increase in both VP and OT release. It thus seems certain that the other candidate is at least a Cl⫺ channel. Although there are some other GABARs, like the GABACR, that are insensitive to BIC but sensitive to PIC, a role for these receptors is highly unlikely since GABAAR is the only type of GABAR that has been reported to exist in NL (Zhang and Jackson, 1993). Since there might be a strychnine-insensitive population of glyRs in NL (Song et al., 2001), it is most likely that the third candidate is still a glyR, but a strychnine-insensitive one. Thus, we suggest that the observed taurine action on basal VP release includes two components, one via GABAARs and another via glyRs, and that two types of glyRs are involved, only one of which is strychnine-sensitive.
Conclusions Our studies further confirmed the mechanism for GABAARs in NL to affect hormone release. Also, we demonstrated that taurine could increase VP/OT hormone release in NL under basal conditions. While the action of taurine on OT release is mediated via GABAAR, the action of taurine on VP release may be mediated via both GABAAR and glyRs. The ability of taurine to increase basal hormone release in NL suggests a depolarizing action of taurine. This partial depolarization of the nerve terminals inactivates Na⫹ channels, thereby blocking propagation of action potentials. It is therefore responsible for the blockade of evoked hormone release from NL by taurine. The action of taurine may be very important in response to system requirements, particularly a reduction of osmotic pressure. These results revealed a new aspect of the interaction between glial cells and nerve terminals in NL, beyond merely a morphological change upon response to physiological challenges.
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Acknowledgments This work was supported by NIH Grant NS09140. The authors thank Dr. C.D. Sladek, T.A. Ponzio, and F. Mercier for suggestions on an earlier draft of the manuscript. References Adams, P.R., Brown, D.A., 1975. Actions of ␥-aminobutyric acid on sympathetic ganglion cells. J Physiol. 250, 85–120. Almarghini, K., Remy, A., Tappaz, M., 1991. Immunocytochemistry of the taurine biosynthesis enzyme, cysteine sulfinate decarboxylase, in the cerebellum — evidence for a glial localization. Neuroscience 43, 111– 119. Beetsch, J.W., Olson, J.E., 1998. Taurine synthesis and cysteine metabolism in cultured rat astrocytes: effects of hyperosmotic exposure. Amer J. Physiol. 274, C866 – 874. Brown, D.A., Marsh, S., 1978. Action of GABA on mammalian peripheral nerves. J. Physiol. 280, 10P. Buijs, R.M., Van Vulpen, E.H.S., Geffard, M., 1987. Ultrastructural localization of GABA in the supraoptic nucleus and neural lobe. Neuroscience 20, 347–355. Decavel, C., Hatton, G.I., 1995. Taurine immunoreactivity in the rat supraoptic necleus: prominent localization in glial cells. J. Comp. Neurol. 354, 13–26. Deleuze, C., Duvoid, A., Hussy, N., 1998. Properties and glial origin of osmotic-dependent release of taurine from the rat supraoptic nucleus. J. Physiol. 507, 463– 471. Dyball, R.E.J., Shaw, F.D., 1978. Inhibition by GABA of hormone release from the neurohypophysis in the rat. J. Physiol. 289, 78 –79P. Fjalland, B., Christensen, J.D., Grell, S., 1987. GABA receptor stimulation increases the release of vasopressin and oxytocin in vitro. Eur. J. Pharmacol. 142, 155–158. Hussy, N., Deleuze, C., Pantaloni, A., Desarmenien, M.G., Moos, F., 1997. Agonist action of taurine on glycine receptors in rat supraoptic magnocellular neurones: possible role in osmoregulation. J. Physiol. 502.3, 609 – 621. Hussy, N., Vanessa, B., Rochette, M., Duvoid, A., Alonso, G., Dayanithi, G., Moos, F.C., 2001. Osmoregulation of vasopressin secretion via activation of neurohypophysial nerve terminals glycine receptors by glial taurine. J. Neurosci. 21 (18), 7110 –7116. Iversen, L.L., Iversen, S.D., Bloom, F.E., 1980. Opiate receptors influence vasopressin release from nerve terminals in rat neurohypophysis. Nature 284, 350 –351. Jackson, M.B., Zhang, S.J., 1995. Action potential propagation and propagation block by GABA in rat posterior pituitary nerve terminals. J. Physiol. 483.3, 597– 611. Jang, I.S., Jeong, H.J., Akaike, N., 2001a. Contribution of Na-K-Cl cotransporter on GABAA receptor-mediated presynaptic depolarization in excitatory nerve terminals. J. Neurosci. 21 (16), 5962–5972. Jang, I.S., Jeong, H.J., Katsurabayashi, S., Akaike, N.,2001b. GABAA receptor in glycinergic nerve terminals and its functional role. Soc. Neurosci. Abstr. 27, Program 498.4.
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Kushe, J., Schmieden, V., Betz, H., 1990. A single amino acid exchange alters the pharmacology of neonatal rat glycine receptors subunit. Neuron. 5, 867– 873. Magnusson, A.M., Meyerson, B.J., 1993. GABA-A agonist muscimol inhibits stimulated vasopressin release in the posterior pituitary of Sprague–Dawley, Wistar, Wistar–Kyoto and spontaneously hypertensive rats. Neuroendocrinology 58, 519 –524. Miyata, S., Matsushima, O., Hatton, G.I., 1997. Taurine in rat posterior pituitary: localization in astrocytes and selective release by hypoosmotic stimulation. J. Comp. Neurol. 381, 513–523. Oertel, W.H., Mugnaini, E., Tappaz, M.L., Weise, V.K., 1982. Central GABAergic innervation of neurointermediate pituitary lobe: biochemical and immunocytochemical study in the rat. Proc. Natl. Acad. Sci. USA. 79, 675– 679. Randle, J.C.R., Renaud, L.P., 1987. Actions of gamma-aminobutyric acid on rat supraoptic nucleus neurosecretory neurones in vitro. J. Physiol. 387, 629 – 647. Saridaki, E., Carter, D.A., Lightman, S.L., 1989. ␥-Aminobutyric acid regulation of neurohypophysial hormone secretion in male and female rats. J. Endocrinol. 121, 343–349. Schlichter, R., Demeneix, B.A., Desarmenien, M., Desaulles, E., Loeffler, J.P., Feltz, P., 1984. Properties of the GABA receptors located on spinal primary afferent neurones and hypophyseal neuroendocrine cells of the rat. Neurosci. Lett. 47, 257–263. Sladek, C.D., Armstrong, W.E., 1987. ␥-Aminobutyric acid antagonist stimulate vasopressin release from organ-cultured hypothalamo-neurohypophyseal explants. Endocrinology 120, 1576 –1580. Song, Z., Itkis, O., Hatton, G.I.,2001. Glycine receptors (glyRs) in rat neurohypophysis: Western blot and functional studies. Soc. Neurosci. Abstr. 27, Program 179.10. Song, Z., Mercier, F., Hatton, G.I., 2000. Immunocytochemical evidence for glycine receptors in both supraoptic nucleus and posterior pituitary: a further role for taurine. Soc. Neurosci. Abstr. 26 (2), 1455. Stern, J.E., Armstrong, W.E., 1995. Electrophysiological differences between oxytocin and vasopressin neurones recorded from female rats in vitro. J. Physiol. 488, 701–708. Taraskevich, P.S., Douglas, W.W., 1982. GABA directly affects electrophysiological properties of pituitary pars intermedia cells. Nature 299, 733–734. Tomiko, S.A., Taraskevich, P.S., Douglas, W.W., 1983. GABA acts directly on cells of pituitary pars intermedia to alter hormone output. Nature 301, 706 –707. Turecek, R., Trussell, L.O., 2001. Presynaptic glycine receptors enhance transmitter release at a mammalian central synapse. Nature 411, 587– 590. Zhang, S.J., Jackson, M., 1993. GABA-activated chloride channels in secretory nerve endings. Science 259, 531–534. Zhang, S.J., Jackson, M., 1995a. Properties of the GABAA receptor of rat posterior pituitary nerve terminals. J. Neurophysiol. 73, 1135–1144. Zhang, S.J., Jackson, M.B., 1995b. GABAA receptor activation and the excitability of nerve terminals in the posterior pituitary. J. Physiol. 483, 583–595. Zingg, H.H., Baertschi, A.J., Dreifuss, J.J., 1979. Action of g-aminobutyric acid on hypothalamo-neurohypophysial axons. Brain Res. 171, 453– 459.
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Taurine and the control of basal hormone release from rat neurohypophysis Zhilin Song and Glenn I. Hatton* Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA Received 27 November 2001; revised 9 January 2003; accepted 16 January 2003
Abstract Pituicytes of pituitary neural lobe are rich in the amino acid taurine, which they release upon hypoosmotic stimulation. As a generally inhibitory amino acid, taurine is thought to activate receptors on neural lobe nerve terminals and exert some control over hormone release. Previous work has shown the presence of glycine and GABAA receptors in neural lobe, both of which have affinity for taurine. Using a perifused explant system, we studied the effects of taurine activation of glycine and GABAA receptors on basal hormone release. Somewhat surprisingly, taurine induced increases in basal release of both vasopressin and oxytocin. Taurine-induced increases in oxytocin release were blocked by bicuculline, suggesting involvement of GABAA receptors. Increases in vasopressin release were not blocked by bicuculline, indicating involvement of receptors other than GABAA. Although combined bicuculline and strychnine, an antagonist at most glycine receptors, also did not block increased vasopressin release, picrotoxin (a Cl⫺ channel blocker) was effective in blocking increases in both vasopressin and oxytocin release. The other receptor(s) involved in taurine actions is postulated to be strychnine-insensitive glycine receptors. Thus, taurine in neural lobe may act via both a GABAA receptor and one or more types of glycine receptors to depolarize nerve terminal membranes under basal conditions. Taurine-induced partial depolarization resulting in Na⫹ channel inactivation is probably responsible for its previously observed inhibition of stimulated hormone release from neural lobe. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Pituitary neural lobe; Pituicytes; Magnocellular hypothalamo-neurohypophysial system; Vasopressin; Oxytocin; GABAA receptor; Glycine receptor
Introduction Taurine, a GABA-like amino acid, is generally regarded as an osmoregulator of the cell. Taurine is present in supraoptic nucleus (SON) of the magnocellular hypothalamoneurohypophysial system (mHNS). In SON, taurine was found predominantly in astrocytes, with traces in occasional dendrites and somata, suggesting uptake by these latter cellular compartments (Decavel and Hatton, 1995). Astrocytes have been shown to express the synthetic enzyme for taurine, cysteine sulfinic acid decarboxylase, and to be capable of rapid taurine synthesis (Almarghini et al., 1991; Beetsch and Olson, 1998). Ultrastructural studies revealed that taurine-rich astrocytes completely engulfed many of the * Corresponding author. Fax: ⫹1-909-787-2967. E-mail address: [email protected] (G.I. Hatton).
axodendritic synaptic contacts in the SON dendritic zone (Decavel and Hatton, 1995), and that astrocytic processes covering the somata frequently contained large amounts of taurine. This suggests that taurine released from astrocytes into the space between the glial and neural elements could attain rather high concentrations at the neural membrane. It was demonstrated that taurine was released from SON glial cells under basal conditions (normo- and hypoosmotic), indicating that taurine may have a tonic inhibitory influence on SON firing, particularly of vasopressin (VP) cells (Deleuze et al., 1998). This strongly suggests that astrocytederived taurine acts as a modulator in the control of hormone release. The involvement of glycine receptors (glyRs) was suggested by the much higher affinity of glyRs for taurine compared with that of GABAA receptors (GABAARs) for taurine in the SON (Hussy et al., 1997; Randle and Renaud, 1987). Physiological studies revealed
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that taurine released from SON glia acts on glyRs to induce neuronal hyperpolarization via increased Cl⫺ conductance, while locally applied strychnine, a selective glyR antagonist, increased electrical activity of VP neurons in normally hydrated rats. This increase was greatly enhanced in waterloaded rats (Hussy et al., 1997). Together, these studies suggest that taurine is tonically released by astrocytes in the SON under normo- to hypoosmotic conditions, and that the amount of taurine released is sufficient to play a role in the overall inhibition of SON neurons. Glial withdrawal during mHNS activation, then, is tantamount to removal of one source of tonic inhibition induced by taurine via glyRs at the soma-dendritic level. Interestingly, in neural lobe (NL) of the mHNS, where axons and terminals originating from SON reside and hormones are released, taurine was found to be rich and exclusively present in pituicytes. Furthermore, taurine was released from NLs upon hypoosmotic stimulation (Miyata et al., 1997). It is reasonable to postulate that taurine may also act directly on the NL axons/terminals to regulate hormone release as it does in the SON. Since the NL is the final site in mHNS at which control of hormone release into the blood can take place, it is important to understand the mechanisms operating at this level. While it is reasonable to suppose that some of the same receptors would be expressed on the terminals as are expressed at the soma-dendritic level of these neurons, it is also possible that they are different. Central GABAergic innervation of NL has been revealed by biochemical and ultrastructural immunocytochemical studies (Buijs et al., 1987; Oertel et al., 1982). Existence of GABAARs on nerve terminals in NL was demonstrated electrophysiologically and blockade of action potential propagation or reduction in amplitude of compound action potentials was achieved by GABAAR activation (Jackson and Zhang, 1995; Zhang and Jackson, 1993, 1995a, 1995b; Zingg et al., 1979). Little is yet known about glyRs in NL. Since taurine at high concentrations can activate GABAARs, taurine’s action on neural membrane in NL could be mediated via either one or both of these two receptors. In the present study, we demonstrated an enhancing effect on basal hormone release as a result of activating GABAARs by GABA and glyRs by glycine. Taurine had effects similar to those of GABA on basal VP and oxytocin (OT) release in NL and its effects were blocked by picrotoxin, a Cl⫺ channel blocker. It is likely that the taurine effects on OT release in NL were mediated via GABAARs, whereas its effects on VP release were mediated via both GABAARs and glyRs.
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the Institutional Animal Care and Use Committee at the University of California, Riverside. Superfusion of NL explants Brains were removed from rats and pituitaries were taken out. Anterior lobe was removed. As much intermediate lobe as possible was removed without damaging the NL. NL explants were perifused individually in closed culture chambers (0.5 mL) at 37°C with oxygenated (95% O2, 5% CO2) ACSF at a rate of 3.75 ml/h. The ACSF consists of the following components (in mM): NaCl 126, NaHPO4 1.3, NaHCO3 26, KCl 5, CaCl2 2.4, MgSO4 1.3, glucose 10, and 3[N-morpholino] propane sulfonic acid buffer 5, pH 7.4. This organic buffer, in combination with the bicarbonate buffer, has been found in our earlier studies to provide better pH stability in long duration experiments than use of the bicarbonate buffer alone. The osmolality of this ACSF is 310 mOsm/l, with an effective osmolality of 300 mOsm/l. To better preserve and prolong tissue viability, ACSF was modified with 0.2 mM ascorbic acid and 0.2 mM thiourea (Stern and Armstrong, 1995). Bacitracin (400 mg/l) was also added to the ACSF to prevent hormone degradation (Sladek and Armstrong, 1987). After 3 h of equilibration, explants were maintained either continuously under ACSF as control or ACSF fortified with different reagents. Outflow was collected individually at 15-min intervals using a fraction collector maintained at 4°C. Data analysis Both VP and OT contents in the perifusate were determined by a sensitive enzyme immunoassay (EIA) kit (Assay Designs, Inc., Ann Arbor, MI). Basal hormone release was calculated for each explant as the mean hormone release during the period just before drug exposure. VP and OT release were expressed as percentage of the basal release for that explant. The results were expressed as mean ⫾ SEM. Analysis of variance (ANOVA) with repeated measures was performed using Sigma Stat in order to evaluate differences in VP and OT release as a result of drug treatment. Subsequent ANOVA and post hoc Student Neuman–Keuls tests were performed to establish specific group differences at individual time points.
Results Taurine effects on VP and OT release
Materials and methods Animals Male Sprague–Dawley rats 45– 60 days old were used in all experiments, and were killed by decapitation. All experimental protocols were done within National Institute of Health guidelines for animal research and were approved by
We evaluated the direct functional impact of taurine on basal hormone release. To determine an optimal concentration, taurine at 1, 10, or 50 mM was administered sequentially to NL explants for 0.5 h each. Since a consistent, near maximal response was achieved at 10 mM (data not shown), this concentration was deemed appropriate and was adopted for subsequent experiments involving taurine. In this and
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subsequent experiments, both VP and OT release were monitored from the same NLs. Taurine significantly increased VP release throughout the treatment period (F ⫽ 26.219, P ⬍ 0.001) (Fig. 1A). Basal VP release for taurine-treated and control groups was 31.8 ⫾ 6.6 and 27.4 ⫾ 4.9 pg/mL, respectively. OT release was also significantly increased by taurine treatment (F ⫽ 22.672, P ⬍ 0.001) (Fig. 1B). Basal OT release for taurine-treated and control groups was 46.3 ⫾ 7.4 and 75.2 ⫾ 17.3 pg/ml, respectively. Glycine effect on VP release Glycine’s effect on basal hormone release was evaluated. After a 3-h equilibration period, NL explants were exposed to either continued control ACSF or to ACSF containing Fig. 2. VP release upon glycine treatment. Glycine at 300 M was applied in one group (Œ). Release results were compared with that of a group that was maintained in control ACSF (F). Glycine treatment significantly increased VP release from neural lobe compared with control group. *P ⬍ 0.05 for mean comparison at individual time points between these two groups. Arrows indicate when glycine actually reached the explants.
300 M glycine. A significant increase in VP release was observed with glycine treatment compared to that of control explants (F ⫽ 36.842, P ⬍ 0.001) (Fig. 2). Basal VP release for glycine-treated and control groups was 17.0 ⫾ 2.2 and 22.5 ⫾ 3.9 pg/ml, respectively. OT release was not monitored in this experiment, since activation of glyR seems to have no apparent effect on basal OT release from related experiments (data not shown). GABA effects on VP and OT release Next we assessed the effect of GABAR activation by GABA on basal hormone release from the NL. Explants were equilibrated for 3 h and then perifused continuously with control ACSF or exposed to ACSF containing 100 M or 1 mM GABA sequentially for 0.5 h each. Results are shown in Fig. 3. As seen in Fig. 3A, GABA treatment significantly increased VP release (F ⫽ 11.490, P ⫽ 0.007). Basal VP release for GABA-treated and control groups was 40.2 ⫾ 6.8 and 45.9 ⫾ 12.8 pg/ml, respectively. Exposure to 1 mM GABA also produced a significant increase in OT release (F ⫽ 10.517, P ⫽ 0.008) (Fig. 3B). Basal OT release for GABA-treated and control groups was 56.5 ⫾ 6.8 and 82.6 ⫾ 7.4 pg/ml, respectively. It appears that GABAR activation in NL can increase basal release of both VP and OT, with the VP response being more sensitive and robust than the OT response. Fig. 1. VP and OT release upon taurine treatment. Taurine at 10 mM was applied in one group (F). Release results were compared with that of control group that was maintained in control ACSF (Œ). Both VP and OT release were monitored. Both VP (A) and OT (B) basal releases were significantly increased by taurine treatment during the entire experiment period (1.5 h). *P ⬍ 0.05 (Student Newman–Keuls test) for mean comparison at individual time points in both (A) and (B). Arrows indicate when taurine actually reached the explants.
Bicuculline and strychnine effects on taurine-induced hormone release Whether the taurine effect on basal hormone release is via GABAAR or glyR activation is unknown. To elucidate this, explants were treated with 10 mM taurine, then taurine
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increase in OT release was due to taurine treatment alone. Together with BIC or a combination of BIC and strychnine, taurine failed to change OT release significantly. Basal OT release for drug-treated and control group was 99.5 ⫾ 11.3 and 106.0 ⫾ 6.1 pg/ml, respectively. Both the VP and the OT responses to taurine treatment showed a slightly longer time lag in this experiment compared to the previous one (cf. Figs. 1 and 4). This was probably due to relatively larger basal VP and OT release in this experiment. Picrotoxin effect on taurine-induced hormone release Although combined BIC and strychnine were not able to block taurine-induced increase in basal VP release
Fig. 3. VP and OT release upon GABA treatment. GABA at 100 M and 1 mM were applied sequentially for 0.5 h each in one group (F). Release results were compared with that of control group that was maintained in continuous ACSF (Œ). (A) GABA at 1 mM significantly increased VP release at both time points (*P ⬍ 0.01 by Student Newman–Keuls test). GABA at 100 M had a tendency to increase VP release. P values for individual time points are as follows: 5, 0.226; 6, 0.097; 7, 0.002; 8, ⬍0.001. (B) GABA at 1 mM increased OT hormone release, but this increase was statistically significant only at the last time point (* P ⬍ 0.05 by Student Newman–Keuls test).
plus 50 M bicuculline (BIC, a GABAAR antagonist), followed by taurine plus a combination of BIC and 10 M strychnine (a glyR antagonist), 0.5 h each. Results are shown in Fig. 4. VP release was elevated by taurine treatment and this effect was not abolished by coapplication of BIC or BIC/strychnine along with taurine (F ⫽ 17.520, P ⫽ 0.002) (Fig. 4A). Basal VP release for drug-treated and control groups was 40.3 ⫾ 5.7 and 36.2 ⫾ 3.6 pg/ml, respectively. For OT release, ANOVA revealed a statistically significant interaction between group and time (F ⫽ 3.487, P ⫽ 0.003) (Fig. 4B). Post hoc Student Newman– Keuls test determined the significance levels for mean comparison at individual time points as follows: 4, P ⬍ 0.001; 5, P ⬍ 0.087; 6, P ⬍ 0.297; 7, P ⬍ 0.605; 8, P ⬍ 0.132. The
Fig. 4. Effect of bicuculline and strychnine on taurine-induced VP and OT release. In one group (F), taurine 10 mM alone was applied first, followed by taurine plus 50 M bicuculline (BIC) and then taurine together with BIC and 10 M strychnine, 0.5 h each. Release results were compared with that of control group that was maintained in control ACSF (Œ). (A) Taurine increased VP hormone release and this effect was not blocked by either BIC alone or BIC plus strychnine (*P ⬍ 0.05 by Student Newman–Keuls test). (B) Taurine at 10 mM significantly increased OT release (*P ⬍ 0.001 by Student Newman–Keuls test). This effect was blocked by BIC alone or BIC plus strychnine.
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rine (probably via both GABA A Rs and strychnineinsensitive glyRs), influence hormone release in NL. In Figs. 5A (VP) and B (OT), ANOVA results for all three groups were as follows: for VP, F ⫽ 0.000247, P ⫽ 1.000; for OT, F ⫽ 0.352, P ⫽ 0.710. Basal VP release (A) for PIC, PIC ⫹ taurine and control groups was 29.0 ⫾ 10.0, 33.2 ⫾ 4.5, and 37.5 ⫾ 4.1 pg/ml, respectively. Basal OT release (B) for PIC, PIC ⫹ taurine and control groups was 42.9 ⫾ 13.7, 59.2 ⫾ 10.6, and 70.2 ⫾ 13.6 pg/ml, respectively. Taurine effect on VP and OT release in Ca2⫹-free perifusion ACSF
Fig. 5. Picrotoxin effect on taurine-induced hormone release. Picrotoxin (PIC 100 M) alone (Œ) or PIC together with 10 mM taurine (■) was included in perifusion medium. Release results from these two groups were compared with that of control group (F) that was maintained continuously in ACSF. PIC alone did not change either VP (A) or OT (B) basal hormone release. In either case (VP or OT), the expected increase in hormone release upon 10 mM taurine treatment was not seen when PIC 100 M was present. Arrows indicate when the drugs actually reached the explants.
(Fig. 4A), it is still possible that taurine effect on VP release was mediated via a combination of glyR and GABAAR, since a strychnine-insensitive population of glyRs might exist in NL (Kushe et al., 1990; Song et al., 2001). To investigate this possibility, picrotoxin (PIC), a specific chloride channel blocker, known to block both GABAAR- and glyR-operated chloride channels, was tested for its ability to block taurine-induced hormone release. PIC (100 M) alone did not change basal release of either hormone (Fig. 5). As predicted, increase in neither VP nor OT release in response to 10 mM taurine treatment was seen when 100 M PIC was present. This result suggests that chloride channels, activated by tau-
To demonstrate Ca2⫹-dependence of taurine effects on release, VP and OT release upon taurine treatment was evaluated in Ca2⫹-free perifusion ACSF. The 2.4 mM CaCl2 · 2H2O in regular ACSF was replaced by 2.4 mM MgCl2 · 6H2O to maintain osmolar and Cl⫺ balance. The explants were equilibrated for 3 h as usual. This procedure also flushed out endogenous extracellular free Ca2⫹. Then taurine at 10 mM was added to the perifusion medium of one group, while control group was continuously maintained in control ACSF. Results are shown in Fig. 6. Taurine failed to induce any increase in either VP (Fig. 6A) or OT (Fig. 6B) hormone release, indicating that extracellular Ca2⫹ is required for taurine to stimulate either VP or OT basal hormone release. ANOVA results were as follows: for VP, F ⫽ 0.203, P ⫽ 0.663; for OT, F ⫽ 1.860, P ⫽ 0.695. Basal VP release (Fig. 6A) for taurine and control groups was 9.0 ⫾ 1.9 and 11.0 ⫾ 2.7 pg/ml, respectively. Basal OT release (Fig. 6B) for taurine and control groups was 113.7 ⫾ 16.5 and 115.1 ⫾ 17.5 pg/ml, respectively.
Discussion Data from the present studies indicate that taurine in the NL increases basal release of both VP and OT. Since taurine is an agonist for both GABAARs and glyRs, it was postulated that taurine could act through GABAARs and/or glyRs to exert its control on hormone release at nerve terminals of the NL. Moreover, we demonstrated the effect of activation of GABAARs or glyRs on basal hormone release. Further studies with receptor antagonists were conducted to elucidate the underlying mechanisms. Role of GABAAR in VP and OT release in NL GABAARs have long been implicated as having a functional role in regulation of hormone release from the NL. The underlying mechanisms involved, however, are not clear, and results from previous studies on hormone release
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Fig. 6. VP and OT release upon taurine treatment in Ca2⫹-free ACSF. Taurine at 10 mM was applied in one group (F). Release results were compared with that of control group that was maintained in control ACSF (Œ). Both VP and OT release were monitored. In either case (VP (A) or OT (B)), the expected increase in hormone release upon 10 mM taurine treatment was not seen when ACSF was free of Ca2⫹. Arrows indicate when taurine actually reached the explants.
in NL were controversial. GABAAR activation by GABA or other agonists like muscimol inhibited intense electrical or excess K⫹ stimulation-induced VP and OT release (Dyball and Shaw, 1978; Magnusson and Meyerson, 1993; Saridaki et al., 1989). Conversely, VP/OT release induced by submaximal electrical stimulation was greatly enhanced by isogauvacin, a GABAAR agonist (Fjalland et al., 1987). However, GABA did not affect spontaneous hormone release in previous studies (Dyball and Shaw, 1978; Iversen et al., 1980). In our current studies, an increase in both VP and OT basal hormone release was observed with GABA treatment of the NL, confirming the opposite effects of GABAAR activation on hormone release in NL under strong stimulation vs basal conditions. Similarly, GABA inhibited stimulated melanocyte stimulating hormone (MSH) release from intermediate lobe (IL), but increased nonstimulated
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MSH release (Schlichter et al., 1984; Tomiko et al., 1983). How does this bipolar effect of GABAAR activation on hormone release occur? One answer comes from electrophysiological analyses in NL terminals. Patch– clamp studies revealed a relatively high intracellular Cl⫺ concentration (⬃20 mM) in nerve terminals of NL, which resulted in a ⫹14-mV membrane depolarization when activated by 50 M GABA (Zhang and Jackson, 1993, 1995a, 1995b). This amount of depolarization did not reach the threshold for Na⫹ channels to generate action potentials but instead inactivated Na⫹ channels, and thus blocked propagation of action potentials down to the nerve terminals (Jackson and Zhang, 1995). The blockade of action potential propagation may contribute to the inhibition of electrically stimulated hormone release by GABAR activation. On the other hand, this amount of depolarization by GABAR activation alone might be big enough to activate some types of voltagesensitive Ca2⫹ channels, leading to a rise of intracellular Ca2⫹ concentration in nerve endings that was responsible for increased basal hormone release. This explanation is supported by recent reports that a weakly depolarizing Cl⫺ current induced by activation of presynaptic GABAARs or glyRs was able to activate Ca2⫹ channels and enhance transmitter release (Jang et al., 2001a, 2001b; Turecek and Trussell, 2001). It is further evidenced by our observation that taurine effects on basal VP and OT release depended on extracellular Ca2⫹. This kind of action on GABAAR activation may also account for the increase of MSH release from IL under basal conditions. However, blockade of action potential propagation by a partial depolarization from GABAAR activation does not address how GABAR activation suppressed excess K⫹-induced hormone release in NL (Hussy et al., 2001). Electrophysiological studies on superior cervical sympathetic ganglion neurons revealed that GABA receptor activation resulted in a depolarization of neuronal membrane and a great reduction in membrane resistance. This action provides a “shunt” to the circuit across neuronal membrane and leads to a reduction in amplitude of orthodromic stimulation-induced spikes or direct stimulation-induced synaptic potentials (Adams and Brown, 1975; Brown and Marsh, 1978). In the adjacent IL, GABAR activation also caused a reduction in spike generation and reduced the amplitude of excess (100 mM) K⫹-induced membrane depolarization via a partial depolarization (Taraskevich and Douglas, 1982). This can account for an increase in MSH release under basal conditions but a suppression of excess K⫹-induced MSH release in IL by GABAR activation. Actions of this sort with GABAR activation may also explain why in NL, GABAAR activation increased basal or submaximal stimulation-induced, but reduced K⫹-stimulated, VP and OT release. Our observation of GABA-induced increased basal hormone release in NL is consistent with these findings and supplies further supporting evidence for the postulated mechanisms of GABAAR action on hormone release in NL.
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Taurine effect on VP and OT release in NL and the role of GABAAR Although it was reported previously that a lower concentration of taurine had no apparent effect on basal hormone release in NL (Hussy et al., 2001), in our current studies, a higher concentration of taurine administered exogenously increased both VP and OT hormone release under basal conditions, mimicking the effect of activating GABAARs by GABA itself. Furthermore, the increase in OT release by taurine was blocked by the GABAAR antagonist bicuculline. This strongly suggests that taurine in NL was capable of altering hormone release via activation of GABAARs. Its effect on hormone release should be similar to that of GABA, that is, to slightly increase basal hormone release, but suppress stimulated hormone release. Taking into account that taurine is abundant in pituicytes and it is released in response to a decrease in osmotic pressure (Miyata et al., 1997), it may be reasonable to postulate that in vivo, taurine could be released in NL in response to a reduction in osmotic pressure, and thus limit stimulated hormone release. What is its relation to system function? The mHNS can be aroused by many different stimuli, such as blood volume loss and emotional changes. These arousing signals are converted into action potentials in magnocellular neurons, which are transmitted to the nerve endings in NL. Depolarization of terminal membrane triggers hormone release in response to these stimuli. The resulting elevation in plasma VP levels could increase water retention by the kidneys and thus decrease systemic osmotic pressure. This decrease in osmotic pressure would lead to taurine release from pituicytes and its subsequent action on nerve terminals would block the further propagation of action potentials down to nerve endings. Taurine would therefore serve as a “shut off” switch for VP/OT hormone release. Involvement of glyRs in taurine regulation of VP/OT hormone release in NL Taurine action in the NL may be mainly mediated via GABAARs (as appears to be the case for OT), but a role for glyRs could not be excluded. First, the existence of glyRs in NL was demonstrated by immunocytochemical studies (Hussy et al., 2001; Song et al., 2000). Western blot studies also showed the existence of glyRs in NL (Song et al., 2001). Since taurine is an agonist at glyRs and the affinity of glyRs for taurine is even higher than that of GABAARs for taurine, it may be that glyRs are important taurine targets in the NL. Second, although the taurine-induced increase in basal OT release was blocked by BIC, that GABAAR antagonist did not block the increase in basal VP release in response to taurine. Hence, there must be at least one other type of receptor involved in regulation of VP release, and the glyR appears to be an ideal candidate. Third, glycine was able to increase basal VP release from NL in our studies. Taken together, this evidence suggests a role for
glyRs in taurine action on nerve terminals of NL. Direct evidence for the involvement of glyRs in taurine action in NL comes from a recent report by Hussy et al. (2001), in which taurine was able to inhibit high K⫹-induced VP release via a strychnine-sensitive glyR. All the evidence above indicates involvement of a strychnine-sensitive glyR in taurine actions, although glyR may have played a much lesser role in taurine regulation of OT release, as shown by our result that taurine-increased OT basal release was blocked by BIC and supported by the fact that much lower expression of glyR was reported in OT nerve endings (Hussy et al., 2001). Since combined treatment with BIC and strychnine did not block (or just partially blocked) taurine-induced increase in basal VP release, there has to be a third component other than GABAAR and strychninesensitive glyRs involved in taurine actions. In one of our experiments, the chloride channel blocker picrotoxin successfully blocked the expected taurine-induced increase in both VP and OT release. It thus seems certain that the other candidate is at least a Cl⫺ channel. Although there are some other GABARs, like the GABACR, that are insensitive to BIC but sensitive to PIC, a role for these receptors is highly unlikely since GABAAR is the only type of GABAR that has been reported to exist in NL (Zhang and Jackson, 1993). Since there might be a strychnine-insensitive population of glyRs in NL (Song et al., 2001), it is most likely that the third candidate is still a glyR, but a strychnine-insensitive one. Thus, we suggest that the observed taurine action on basal VP release includes two components, one via GABAARs and another via glyRs, and that two types of glyRs are involved, only one of which is strychnine-sensitive.
Conclusions Our studies further confirmed the mechanism for GABAARs in NL to affect hormone release. Also, we demonstrated that taurine could increase VP/OT hormone release in NL under basal conditions. While the action of taurine on OT release is mediated via GABAAR, the action of taurine on VP release may be mediated via both GABAAR and glyRs. The ability of taurine to increase basal hormone release in NL suggests a depolarizing action of taurine. This partial depolarization of the nerve terminals inactivates Na⫹ channels, thereby blocking propagation of action potentials. It is therefore responsible for the blockade of evoked hormone release from NL by taurine. The action of taurine may be very important in response to system requirements, particularly a reduction of osmotic pressure. These results revealed a new aspect of the interaction between glial cells and nerve terminals in NL, beyond merely a morphological change upon response to physiological challenges.
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