Adenosine trisphosphate appears to act via different receptors in terminals versus somata of the hypothalamic neurohypophysial system.

ATP-induced ionic currents were investigated in isolated terminals and somata of the hypothalamic neurohypophysial system (HNS). Both terminals and somata showed inward rectification of the ATP-induced currents and reversal near 0 mV. In terminals, ATP dose-dependently evoked an inactivating, inward current. However, in hypothalamic somata, ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence (EC(50) of 50 µm in somata versus 9.6 µm in terminals). The ATP-induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a purinergic receptor (P2XR). However, the suramin inhibition was significantly different in the two HNS compartments (IC(50) of 3.6 µm in somata versus 11.6 µm in terminals). Also, both HNS compartments show significantly different responses to the purinergic receptor agonists: ATP-γ-S and benzoyl-benzoyl-ATP. Finally, there was an initial desensitisation to ATP upon successive stimulations in the terminals, which was not observed in the somata. These differences in EC(50) , inactivation, desensitisation and agonist sensitivity in terminals versus somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurones. Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4 and 7 (but not 6) have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and oxytocin terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals versus somata of these central nervous system neurones.

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.

Structural difference between heteromeric somatic and homomeric axonal glycine receptors in the hypothalamo-neurohypophysial system.

Glycine receptors are ionotropic receptors formed by either the homomeric assembly of ligand-binding alpha subunits or the heteromeric combination of an alpha subunit and the auxiliary beta subunit. Glycine receptors in the brain are found at either pre- or post-synaptic sites. Rat supraoptic nucleus neurons express glycine receptors on the membrane of both their soma and dendrites within the supraoptic nucleus, and their axon terminals in the neurohypophysis. Taking advantage of the well-separated cellular compartments of this system, we correlated the structural properties of the receptors to their subcellular localization. Immunohistochemical study using the generic mAb4a antibody revealed that somatodendritic receptors were clustered, whereas axonal glycine receptors showed a more diffuse distribution. This was paralleled by the presence of clusters of the glycine receptor aggregating protein gephyrin in the supraoptic nucleus and its complete absence in the neurohypophysis. Moreover, another antibody recognizing the alpha1/alpha2 subunits similarly labeled the axonal glycine receptors, but did not recognize the somatodendritic receptor clusters of supraoptic nucleus neurons, indicative of structural differences between somatic and axonal glycine receptors. Furthermore, the subunits composing the somatic and axonal receptors have different molecular weight. Functional study further differentiated the two types of glycine receptors on the basis of their sensitivity to picrotoxin, identifying somatic receptors as alpha/beta heteromers, and axonal receptors as alpha homomers. These results indicate that targeting of glycine receptors to axonal or somatodendritic compartment is directly related to their subunit composition, and set the hypothalamo-neurohypophysial system as an excellent model to study the mechanisms of targeting of proteins to various neuronal cellular compartments.

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.

Age-related modifications of the morphological organization of pituicytes are associated with alteration of the GABAergic and dopaminergic innervation afferent to the neurohypophysial lobe.

Ageing is known to induce a marked activation of astrocytes within various regions of the central nervous system. To date, the age-related factors responsible for these modifications are unknown. The neural lobe of the hypophysis (NL) is a particular brain region which does not contain neurons but does contain specialized astrocytes, called pituicytes, and numerous terminals of afferent axons, including (i) peptidergic neurohypophysial axons which terminate on the NL blood vessels, and (ii) axons containing both gamma amino-butyric acid (GABA) and dopamine (DA) which form contacts with pituicytes. Because evidence has recently been provided that GABA signalling mediates the morphological organization of astrocytes, the present study was designed to determine whether modifications of pituicytes during ageing were associated with modifications of the GABAergic axons innervating the NL. We show here that, in adult rats, GABA/DA axons form preferential synaptic-like contacts with pituicytes which express both GABAA and D2 dopamine receptors. We then show that, during ageing, pituicytes undergo dramatic modifications of their morphology, correlatively with marked modifications of the GABA/DA fibres innervating the NL. Lastly, in vitro experiments indicate that modifications of the morphology of pituicytes similar to those observed during ageing were obtained by incubating isolated NL of adult rats with a GABAA receptor agonist and/or a D2 dopamine receptor antagonist, whereas inverse modifications were observed when NL of aged rats were incubated with a GABAA receptor antagonist and a D2 dopamine receptor agonist. Taken together, these data suggest that the age-related morphological changes of pituicytes result from the alteration of the GABA/DAergic innervation of the NL.

Osmoregulation of vasopressin secretion via activation of neurohypophysial nerve terminals glycine receptors by glial taurine.

Osmotic regulation of supraoptic nucleus (SON) neuron activity depends in part on activation of neuronal glycine receptors (GlyRs), most probably by taurine released from adjacent astrocytes. In the neurohypophysis in which the axons of SON neurons terminate, taurine is also concentrated in and osmo-dependently released by pituicytes, the specialized glial cells ensheathing nerve terminals. We now show that taurine release from isolated neurohypophyses is enhanced by hypo-osmotic and decreased by hyper-osmotic stimulation. The high osmosensitivity is shown by the significant increase on only 3.3% reduction in osmolarity. Inhibition of taurine release by 5-nitro-2-(3-phenylpropylamino)benzoic acid, niflumic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid suggests the involvement of volume-sensitive anion channels. On purified neurohypophysial nerve endings, activation of strychnine-sensitive GlyRs by taurine or glycine primarily inhibits the high K(+)-induced rise in [Ca(2+)](i) and subsequent release of vasopressin. Expression of GlyRs in vasopressin and oxytocin terminals is confirmed by immunohistochemistry. Their implication in the osmoregulation of neurohormone secretion was assessed on isolated whole neurohypophyses. A 6.6% hypo-osmotic stimulus reduces by half the depolarization-evoked vasopressin secretion, an inhibition totally prevented by strychnine. Most importantly, depletion of taurine by a taurine transport inhibitor also abolishes the osmo-dependent inhibition of vasopressin release. Therefore, in the neurohypophysis, an osmoregulatory system involving pituicytes, taurine, and GlyRs is operating to control Ca(2+) influx in and neurohormone release from nerve terminals. This elucidates the functional role of glial taurine in the neurohypophysis, reveals the expression of GlyRs on axon terminals, and further defines the role of glial cells in the regulation of neuroendocrine function.

High voltage-activated Ca2+ currents in rat supraoptic neurones: biophysical properties and expression of the various channel alpha1 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 alpha1 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

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 [(3)H]-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, ClC(3), phospholemman and VDAC(1), and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC(2). 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.

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.

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.

Vasopressin regularizes the phasic firing pattern of rat hypothalamic magnocellular vasopressin neurons.

Vasopressin (AVP) magnocellular neurons of hypothalamic nuclei express specific phasic firing (successive periods of activity and silence), which conditions the mode of neurohypophyseal vasopression release. In situations favoring plasmatic secretion of AVP, the hormone is also released at the somatodendritic level, at which it is believed to modulate the activity of AVP neurons. We investigated the nature of this autocontrol by testing the effects of juxtamembrane applications of AVP on the extracellular activity of presumed AVP neurons in paraventricular and supraoptic nuclei of anesthetized rats. AVP had three effects depending on the initial firing pattern: (1) excitation of faintly active neurons (periods of activity of <10 sec), which acquired or reinforced their phasic pattern; (2) inhibition of quasi-continuously active neurons (periods of silences of <10 sec), which became clearly phasic; and (3) no effect on neurons already showing an intermediate phasic pattern (active and silent periods of 10-30 sec). Consequently, AVP application resulted in a narrower range of activity patterns of the population of AVP neurons, with a Gaussian distribution centered around a mode of 57% of time in activity, indicating a homogenization of the firing pattern. The resulting phasic pattern had characteristics close to those established previously for optimal release of AVP from neurohypophyseal endings. These results suggest a new role for AVP as an optimizing factor that would foster the population of AVP neurons to discharge with a phasic pattern known to be most efficient for hormone release.

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 (> or = 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.

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 100 microM glycine), but were unaffected by the GABAA antagonist gabazine (1-3 microM). GABA-activated currents were reversibly antagonized by 3 microM gabazine, but not by strychnine (up to 1 microM). 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 microM. 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 nM) 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.

NMDA receptor properties in rat supraoptic magnocellular neurons: characterization and postnatal development.

Hypothalamo-neurohypophysial magnocellular neurons display specific electrical activities in relation to the mode of release of their hormonal content (vasopressin or oxytocin). These activities are under strong glutamatergic excitatory control. The implication of NMDA receptors in the control of vasopressinergic and oxytocinergic neurons is still a matter of debate. We here report the first detailed characterization of functional properties of NMDA receptors in voltage-clamped magnocellular neurons acutely dissociated from the supraoptic nucleus. All cells responded to NMDA with currents that reversed polarity around 0 mV and were inhibited by D-2-amino-5-phosphonovalerate (D-APV) and by 100 microM extracellular Mg2+ (at -80 mV). Sensitivity to the co-agonist glycine (EC50, 2 microM) was low compared with most other neuronal preparations. The receptors displayed low sensitivity to ifenprodil, were insensitive to glycine-independent potentiation by spermine, and had a unitary conductance of 50 pS. No evidence was found for two distinct cell populations, suggesting that oxytocinergic and vasopressinergic neurons express similar NMDA receptors. Characterization of NMDA receptors at different postnatal ages revealed a transient increase in density of NMDA currents during the second postnatal week. This was accompanied by a specific decrease in sensitivity to D-APV, with no change in NMDA sensitivity or any other properties studied. Supraoptic NMDA receptors thus present characteristics that strikingly resemble those of reconstituted receptors composed of NR1 and NR2A subunits. Understanding the functional significance of the development of NMDA receptors in the supraoptic nucleus will require further knowledge about the maturation of neuronal excitability, synaptic connections and neurohormone release mechanisms.

D2, D3, and D4 dopamine receptors couple to G protein-regulated potassium channels in Xenopus oocytes.

Human D2, D3, D4 and dopamine receptors were individually coexpressed in Xenopus oocytes with a G protein-regulated inwardly rectifying potassium channel (GIRK1). At -100 mV in 96 mM potassium, dopamine (0.1-100 nM) evoked an inward current; the current showed inward rectification, reversed polarity at 0 mV, and was blocked by barium (50% inhibition by 10 microM). The concentrations of dopamine activating 50% of the maximal current (EC50) were not different (2-4 nM) for D2, D3, and D4 receptors, but the maximal current was 3-fold larger for D2 and D4 than for D3 receptors. Dopamine evoked reproducible inward currents at D2 and D4 receptors when applied repeatedly, but second responses could not be observed in oocytes expressing D3 receptors. 7-Hydroxy-N,N-di-n-propyl-2-aminotetralin mimicked the effect of dopamine (EC50 of approximately 2, approximately 3, and approximately 19 nM at D2, D3, and D4, respectively). (-) Sulpiride reversibly blocked the dopamine-induced current with IC50 values of 5, 300, and 2000 nM for D2, D3, and D4 receptors, respectively. Dopamine was ineffective in oocytes injected 2 hr previously with pertussis toxin. We concluded that all three D2-like dopamine receptors share the potential to activate inwardly rectifying potassium channels.

Functional properties of a cloned 5-hydroxytryptamine ionotropic receptor subunit: comparison with native mouse receptors.

1. A comparative study of the whole-cell and single-channel properties of cloned and native mouse 5-hydroxytryptamine ionotropic receptors (5-HT3) was undertaken using mammalian cell lines expressing the cloned 5-HT3 receptor subunit A (5-HT3R-A), superior cervical ganglia (SCG) neurones and N1E-115 cells. 2. No pharmacological difference was found in the sensitivity to the agonists 5-HT and 2-methyl-5-HT, or to the antagonists d-tubocurare and 3-tropanyl-3,5-dichlorobenzoate (MDL-72222). 3. Current-voltage (I-V) relationships of whole-cell currents showed inward rectification in the three preparations. Rectification was stronger both in cells expressing the 5-HT3R-A subunit and in N1E-115 cells when compared with SCG neurones. 4. No clear openings could be resolved in 5-HT-activated currents in patches excised from cells expressing the 5-HT3R-A subunit or N1E-115 cells. Current fluctuation analysis of whole-cell and excised-patch records revealed a slope conductance of 0.4-0.6 pS in both preparations. Current-voltage relationships of these channels showed strong rectification that fully accounted for the whole-cell voltage dependence. 5. In contrast, single channels of about 10 pS were activated by 5-HT in patches excised from SCG neurones. The weak voltage dependence of their conductance did not account completely for the rectification of whole-cell currents. A lower unitary conductance (3.4 pS) was inferred from whole-cell noise analysis. 6. We conclude that the receptor expressed from the cloned cDNA is indistinguishable from the 5-HT3 receptor of N1E-115 cells, suggesting an identical structure for these two receptors. The higher conductance and different voltage dependence of the 5-HT3 receptor in SCG neurones might indicate the participation of an additional subunit in the structure of native ganglionic 5-HT3 receptors. Homo-oligomeric 5-HT3R-A channels may also be present as suggested by the lower conductance estimated by whole-cell noise analysis.

A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP.

Extracellular ATP exerts its effects through P2 purinoceptors: these are ligand-gated ion channels (P2x) or G-protein-coupled receptors (P2Y, P2U). ATP at P2x receptors mediates synaptic transmission between neurons and from neurons to smooth muscle, being responsible, for example, for sympathetic vasoconstriction in small arteries and arterioles. We have now cloned a complementary DNA encoding the P2x receptor from rat vas deferens and expressed it in Xenopus oocytes and mammalian cells. ATP activates a cation-selective ion channel with relatively high calcium permeability. Structural predictions suggest that the protein (399 amino acids long) is mostly extracellular and contains only two transmembrane domains plus a pore-forming motif which resembles that of potassium channels. The P2x receptor thus defines a new family of ligand-gated ion channels.

Organization of the mouse 5-HT3 receptor gene and functional expression of two splice variants.

The structure of the mouse 5-HT3 receptor gene, 5-HT3R-A, is most similar to nicotinic acetylcholine receptor (nAChR) genes, in particular to the gene encoding the neuronal nAChR subunit alpha 7. These genes share among other things the location of three adjacent introns, suggesting that 5-HT3R-A and nAChR genes arose from a common precursor gene. The alternative use of two adjacent splice acceptor sites in intron 8 creates, in addition to the original 5-HT3R-A cDNA (5-HT3R-AL), a shorter isoform (5-HT3R-AS) which lacks six codons in the segment that translates into the major intracellular domain. This splice consensus sequence is not found in human genomic DNA. In mouse, we demonstrate by RNAse protection assay that 5-HT3R-AS mRNA is approximately 5 times more abundant than 5-HT3R-AL mRNA in both neuroblastoma cell lines and neuronal tissues. We used the Semliki Forest virus expression system for electrophysiological characterization of 5-HT3R-AS and 5-HT3R-AL in mammalian cells. No differences in electrophysiological characteristics, such as voltage dependence, desensitization kinetics, or unitary conductance were found between homomeric 5-HT3R-AS and 5-HT3R-AL receptors. Their properties are very similar to those of 5-HT3 receptors in mouse neuroblastoma cell lines.

Agonist and antagonist effects of nicotine on chick neuronal nicotinic receptors are defined by alpha and beta subunits.

1. Functional neuronal nicotinic receptors were reconstituted in Xenopus oocytes by the nuclear injection of different combinations of chick and rat cDNAs encoding alpha and beta subunits. The pharmacology of these nicotinic receptors was investigated using two-electrode voltage clamp. 2. The sensitivity of the chick alpha 3/beta 2, alpha 3/beta 4, and alpha 4/beta 2 receptors to acetylcholine (ACh) and neuronal bungarotoxin differed markedly, indicating that both subunits contribute to the pharmacological properties of the receptors. 3. Nicotine acted as an agonist on the chick alpha 3/beta 4 and alpha 4/beta 2 receptors and rat alpha 3/beta 2 receptor. In contrast, nicotine (at concentrations > 3 microM) was only a weak partial agonist of the chick alpha 3/beta 2 receptor. Moreover, nicotine coapplied with 3 microM ACh on the chick alpha 3/beta 2 receptor acted as a potent competitive antagonist, with an IC50 of 0.43 microM. No antagonist effect of nicotine could be revealed on the other nicotinic receptors. 4. The effect of nicotine was tested on hybrid receptors obtained by coinjection of chick and rat cDNAs encoding the alpha 3 and beta 2 subunits (yielding the rat alpha 3/chick beta 2 and chick alpha 3/rat beta 2 receptors). Nicotine (10 microM) strongly inhibited both hybrid receptors. 5. Chimeric subunits were constructed by exchanging a segment located in the extracellular N-termini of chick alpha 3 and alpha 4 subunits and chick alpha 3 and rat alpha 3 subunits. These subunits were coexpressed in oocytes with chick or rat beta 2 subunits. The effect of nicotine on these receptors pointed to the importance of a 15 amino acid stretch located 3' of the first transmembrane segment in the determination of the agonist and antagonist action of nicotine. 6. Within this 15 amino acid segment, a single residue differs in chick and rat alpha 3 subunits, at position 198, within the ligand binding site of alpha subunits. Gln198 of the rat alpha 3 subunit was replaced by Thr as found in the chick alpha 3 subunit, using site-directed mutagenesis. The mutant subunit was coexpressed with the rat beta 2 subunit, yielding receptors which were inhibited by nicotine. 7. It is concluded that both alpha and beta subunits not only alter considerably the sensitivity of the nicotinic receptor to agonists and antagonists, but also can turn an agonist into a potent antagonist of one receptor subtype.(ABSTRACT TRUNCATED AT 400 WORDS)