Nicolas Hussy

Agonist action of taurine on glycine receptors in rat supraoptic magnocellular neurones: possible role in osmoregulation

1 To evaluate the implication of taurine in the physiology of supraoptic neurones, we (i) investigated the agonist properties of taurine on glycine and GABAA receptors of supraoptic magnocellular neurones acutely dissociated from adult rats, using whole‐cell voltage clamp, (ii) studied the effects of taurine and strychnine in vivo by extracellular recordings of supraoptic vasopressin neurones in anaesthetized rats, and (iii) measured the osmolarity‐dependent release of endogenous taurine from isolated supraoptic nuclei by HPLC. 2 GABA, glycine and taurine evoked rapidly activating currents that all reversed close to the equilibrium potential for Cl−, indicating activation of Cl− selective channels. Glycine‐activated currents were reversibly blocked by strychnine (IC50 of 35 nM with 100μm glycine), but were unaffected by the GABAA antagonist gabazine (1–3 μm). GABA‐activated currents were reversibly antagonized by 3 μm gabazine, but not by strychnine (up to 1 μm). 3 Responses to 1 mm taurine were blocked by strychnine but not by gabazine and showed no additivity with glycine‐induced currents, indicating selective activation of glycine receptors. Responses to 10 mm taurine were partially antagonized by gabazine, the residual current being blocked by strychnine. Thus, taurine is also a weak agonist of GABAA receptors. 4 In the presence of gabazine, taurine activated glycine receptors with an EC50 of 406 μm. Taurine activated at most 70% of maximal glycine currents, suggesting that it is a partial agonist of glycine receptors. 5 In vivo, locally applied strychnine (300 mm) increased and taurine (1 mm) decreased the basal electrical activity of vasopressin neurones in normally hydrated rats. The effect of strychnine was markedly more pronounced in water‐loaded rats. 6 Taurine, which is concentrated in supraoptic glial cells, could be released from isolated supraoptic nuclei upon hyposmotic stimulation. Decreases in osmolarity of 15 and 30% specifically enhanced basal release of taurine by 42 and 124%, respectively. 7 We conclude that supraoptic neurones express high amounts of glycine receptors, of which taurine may be regarded as a major natural agonist. We postulate that taurine, which can be released in hyposmotic situations, acts on glycine receptors to exert an inhibitory control on magnocellular neurones during alterations of body fluid homeostasis, implicating an active participation of glial cells in this neuroendocrine regulatory loop.

Osmotic regulation of neuronal activity: a new role for taurine and glial cells in a hypothalamic neuroendocrine structure

Maintenance of osmotic pressure is a primary regulatory process essential for normal cell function. The osmolarity of extracellular fluids is regulated by modifying the intake and excretion of salts and water. A major component of this regulatory process is the neuroendocrine hypothalamo-neurohypophysial system, which consists of neurons located in the paraventricular and supraoptic nuclei. These neurons synthesize the neurohormones vasopressin and oxytocin and release them in the blood circulation. We here review the mechanisms responsible for the osmoregulation of the activity of these neurons. Notably, the osmosensitivity of the supraoptic nucleus is described including the recent data that suggests an important participation of taurine in the transmission of the osmotic information. Taurine is an amino acid mainly known for its involvement in cell volume regulation, as it is one of the major inorganic osmolytes used by cells to compensate for changes in extracellular osmolarity. In the supraoptic nucleus, taurine is highly concentrated in astrocytes, and released in an osmodependent manner through volume-sensitive anion channels. Via its agonist action on neuronal glycine receptors, taurine is likely to contribute to the inhibition of neuronal activity induced by hypotonic stimuli. This inhibitory influence would complement the intrinsic osmosensitivity of supraoptic neurons, mediated by excitatory mechanoreceptors activated under hypertonic conditions. These observations extend the role of taurine from the regulation of cell volume to that of the whole body fluid balance. They also point to a new role of supraoptic glial cells as active components in a neuroendocrine regulatory loop.

Properties and glial origin of osmotic-dependent release of taurine from the rat supraoptic nucleus

1 Taurine, prominently concentrated in glial cells in the supraoptic nucleus (SON), is probably involved in the inhibition of SON vasopressin neurones by peripheral hypotonic stimulus, via activation of neuronal glycine receptors. We report here the properties and origin of the osmolarity‐dependent release of preloaded [3H]taurine from isolated whole SO nuclei. 2 Hyposmotic medium induced a rapid, reversible and dose‐dependent increase in taurine release. Release showed a high sensitivity to osmotic change, with a significant enhancement with less than a 5 % decrease in osmolarity. Hyperosmotic stimulus decreased basal release. 3 Evoked release was independent of extracellular Ca2+ and Na+, and was blocked by the Cl− channel blockers DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid) and DPC (N‐phenylanthranilic acid), suggesting a diffusion process through volume‐sensitive Cl− channels. 4 Evoked release was transient for large osmotic reductions (≥ 15 %), probably reflecting regulatory volume decrease (RVD). However, it was sustained for smaller changes, suggesting that taurine release induced by physiological variations in osmolarity is not linked to RVD. 5 Basal and evoked release were strongly inhibited by an incubation of the tissue with the glia‐specific toxin fluorocitrate, but were unaffected by a neurotoxic treatment with NMDA, demonstrating the glial origin of the release of taurine in the SON. 6 The high osmosensitivity of taurine release suggests an important role in the osmoregulation of the SON function. These results strengthen the notion of an implication of taurine and glial cells in the regulation of the whole‐body fluid balance through the modulation of vasopressin release.

Pharmacological characterization of volume-sensitive, taurine permeable anion channels in rat supraoptic glial cells

To characterize the volume‐sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [3H]‐taurine efflux from acutely isolated SON. Taurine release induced by hypotonic stimulus (250 mosmol l−1) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, confirming the lack of implication of the transporter. The osmodependent release of taurine was blocked by a variety of Cl− channel inhibitors with the order of potency: NPPB>niflumic acid>DPC>DIDS>ATP. On the other hand, release of taurine was only weakly affected by other compounds (dideoxyforskolin, 4‐bromophenacyl bromide, mibefradil) known to block volume‐activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels. Although the molecular identity of volume‐sensitive anion channels is not firmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC3, phospholemman and VDAC1, and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC2. In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological profile, suggesting the expression of a particular type or subtype of volume‐sensitive anion channel, which is likely to be formed by yet unidentified proteins.

Tyrosine phosphorylation modulates the osmosensitivity of volume‐dependent taurine efflux from glial cells in the rat supraoptic nucleus

1 In the supraoptic nucleus, taurine, selectively released in an osmodependent manner by glial cells through volume‐sensitive anion channels, is likely to inhibit neuronal activity as part of the osmoregulation of vasopressin release. We investigated the involvement of various kinases in the activation of taurine efflux by measuring [3H]taurine release from rat acutely isolated supraoptic nuclei. 2 The protein tyrosine kinase inhibitors genistein and tyrphostin B44 specifically reduced, but did not suppress, both the basal release of taurine and that evoked by a hypotonic stimulus. Inhibition of tyrosine phosphatase by orthovanadate had the opposite effect. 3 The tyrosine kinase and phosphatase inhibitors shifted the relationship between taurine release and medium osmolarity in opposite directions, suggesting that tyrosine phosphorylation modulates the osmosensitivity of taurine release, but is not necessary for its activation. 4 Genistein also increased the amplitude of the decay of the release observed during prolonged hypotonic stimulation. Potentiation of taurine release by tyrosine kinases could serve to maintain a high level of taurine release in spite of cell volume regulation. 5 Taurine release was unaffected by inhibitors and/or activators of PKA, PKC, MEK and Rho kinase. 6 Our results demonstrate a unique regulation by protein tyrosine kinase of the osmosensitivity of taurine efflux in supraoptic astrocytes. This points to the presence of specific volume‐dependent anion channels in these cells, or to a specific activation mechanism or regulatory properties. This may relate to the particular role of the osmodependent release of taurine in this structure in the osmoregulation of neuronal activity.

Zinc is both an intracellular and extracellular regulator of KATP channel function

Extracellular Zn2+ has been identified as an activator of pancreatic KATP channels. We further examined the action of Zn2+ on recombinant KATP channels formed with the inward rectifier K+ channel subunit Kir6.2 associated with either the pancreatic/neuronal sulphonylurea receptor 1 (SUR1) subunit or the cardiac SUR2A subunit. Zn2+, applied at either the extracellular or intracellular side of the membrane appeared as a potent, reversible activator of KATP channels. External Zn2+, at micromolar concentrations, activated SUR1/Kir6.2 but induced a small inhibition of SUR2A/Kir6.2 channels. Cytosolic Zn2+ dose‐dependently stimulated both SUR1/Kir6.2 and SUR2A/Kir6.2 channels, with half‐maximal effects at 1.8 and 60 μm, respectively, but it did not affect the Kir6.2 subunit expressed alone. These observations point to an action of both external and internal Zn2+ on the SUR subunit. Effects of internal Zn2+ were not due to Zn2+ leaking out, since they were unaffected by the presence of a Zn2+ chelator on the external side. Similarly, internal chelators did not affect activation by external Zn2+. Therefore, Zn2+ is an endogenous KATP channel opener being active on both sides of the membrane, with potentially distinct sites of action located on the SUR subunit. These findings uncover a novel regulatory pathway targeting KATP channels, and suggest a new role for Zn2+ as an intracellular signalling molecule.

High Voltage-Activated Ca2+ Currents in Rat Supraoptic Neurones: Biophysical Properties and Expression of the Various Channel α1 Subunits

The diversity of Ca2+ currents was studied in voltage‐clamped acutely dissociated neurones from the rat supraoptic nucleus (SON), and the expression of the various corresponding pore‐forming α1 subunits determined by immunohistochemistry. We observed the presence of all high voltage‐activated L‐, N‐, P/Q‐ and R‐type currents. We did not observe low‐voltage‐activated T‐type current. The multimodal current/voltage relationships of L‐ and R‐type currents indicated further heterogeneity within these current types, each exhibiting two components that differed by a high (−20 mV) and a lower (−40 mV) threshold potential of activation. L‐ and R‐type currents were fast activating and showed time‐dependent inactivation, conversely to N‐ and P/Q‐type currents, which activated more slowly and did not inactivate. The immunocytochemical staining indicated that the soma and proximal dendrites of SON neurones were immunoreactive for Cav1.2, Cav1.3 (forming L‐type channels), Cav2.1 (P/Q‐type), Cav2.2 (N‐type) and Cav2.3 subunits (R‐type). Each subunit exhibited further specificity in its distribution throughout the nucleus, and we particularly observed strong immunostaining of Cav1.3 and Cav2.3 subunits within the dendritic zone of the SON. These data show a high heterogeneity of Ca2+ channels in SON. neurones, both in their functional properties and cellular distribution. The lower threshold and rapidly activating L‐ and R‐type currents should underlie major Ca2+ entry during action potentials, while the slower and higher threshold N‐ and P/Q‐type currents should be preferentially recruited during burst activity. It will be of key interest to determine their respective role in the numerous Ca2+‐dependent events that control the activity and physiology of SON neurones

Extrasynaptic localization of glycine receptors in the rat supraoptic nucleus: Further evidence for their involvement in glia-to-neuron communication

Neurons of the rat supraoptic nucleus (SON) express glycine receptors (GlyRs), which are implicated in the osmoregulation of neuronal activity. The endogenous agonist of the receptors has been postulated to be taurine, shown to be released from astrocytes. We here provide additional pieces of evidence supporting the absence of functional glycinergic synapses in the SON. First, we show that blockade of GlyRs with strychnine has no effect on either the amplitude or frequency of miniature inhibitory postsynaptic currents recorded in SON neurons, whereas they were all suppressed by the GABA(A) antagonist gabazine. Then, double immunostaining of sections with presynaptic markers and either GlyR or GABA(A) receptor (GABA(A)R) antibodies indicates that, in contrast with GABA(A)Rs, most GlyR membrane clusters are not localized facing presynaptic terminals, indicative of their extrasynaptic localization. Moreover, we found a striking anatomical association between SON GlyR clusters and glial fibrillary acidic protein (GFAP)-positive astroglial processes, which contain high levels of taurine. This type of correlation is specific to GlyRs, since GABA(A)R clusters show no association with GFAP-positive structures. These results substantiate and strengthen the concept of extrasynaptic GlyRs mediating a paracrine communication between astrocytes and neurons in the SON.

Chapter 8 Glial cells in the hypothalamo-neurohypophysial system: key elements of the regulation of neuronal electrical and secretory activity

Publisher Summary This chapter discusses the present knowledge of the role of glial cells in the regulation of neuronal activity in the Hypothalamo-Neurohypophysial System (HNS) and the general organization of the system, as the specific anatomical relationships between glia and neurons are directly correlated to the functional properties of the system. The HNS consists of two neuronal populations that synthesize the neurohormones vasopressin (VP) or oxytocin (OT). The cell bodies of these magnocellular neurons are mainly localized in the hypothalamic supraoptic (SON) and paraventrieular nuclei (PVN), although many of them can also be found as accessory groups of neurons scattered between the two nuclei. Three main pieces of evidence provide the basis of the present understanding of the physiological implication of glial cells in the control of neuroendocrine function in the HNS. The HNS is well known for the activity dependent plasticity of the morphological relationships between neurons and glial cells. This mainly results in long-term changes in the functional properties of the neuronal network, by modifying the well-established functions of glial cells, such as regulation of ionic composition or uptake of neurotransmitters, and by regulating other less classical neuron–glia interactions just as those involving release of neuroactive substances by glial cells.

Synergistic activation of astrocytes by atp and norepinephrine in the rat supraoptic nucleus

Supraoptic nucleus (SON) neurons receive a dense innervation from noradrenergic fibers, the activity of which stimulates vasopressin (VP) and oxytocin (OT) release, notably during homeostatic regulation of blood pressure and volume. This regulation is known to involve the co-release of norepinephrine (NE) and ATP, which act in synergy to stimulate Ca(2+) increase in SON neurons and to enhance release of VP and OT from hypothalamo-neurohypophysial explants. We here demonstrate that both ATP and NE also trigger transient intracellular Ca(2+) rise in rat SON astrocytes, the two agonists showing a synergistic action similarly to what has been reported in SON neurons. The responses to both agonists are not or are only moderately affected after blockade of neuronal activity by tetrodotoxin, or of neurotransmitter release by removal of extracellular Ca(2+), suggesting that the receptors involved are located on the astrocytes themselves. ATP acts via P2Y(1) receptors, as indicated by the pharmacological profile of Ca(2+) responses and the strong immunolabeling for this receptor in SON astrocytes. Responses to NE involve both alpha and beta adrenergic receptors, the latter showing a permissive role on the former. These results point to further implication of SON astrocytes in the regulation of VP and OT secretion, and suggest that they are potentially important elements participating in all regulatory processes of hypothalamo-neurohypophysial function that involve activation of noradrenergic pathways.