Neurohypophysial Nerve Terminals Research Articles

Activity-dependent depression of neuronal sodium channels by the general anaesthetic isoflurane

The mechanisms by which volatile anaesthetics such as isoflurane alter neuronal function are poorly understood, in particular their presynaptic mechanisms. Presynaptic voltage-gated sodium channels (Na(v)) have been implicated as a target for anaesthetic inhibition of neurotransmitter release. We hypothesize that state-dependent interactions of isoflurane with Na(v) lead to increased inhibition of Na(+) current (I(Na)) during periods of high-frequency neuronal activity. The electrophysiological effects of isoflurane, at concentrations equivalent to those used clinically, were measured on recombinant brain-type Na(v)1.2 expressed in ND7/23 neuroblastoma cells and on endogenous Na(v) in isolated rat neurohypophysial nerve terminals. Rate constants determined from experiments on the recombinant channel were used in a simple model of Na(v) gating. At resting membrane potentials, isoflurane depressed peak I(Na) and shifted steady-state inactivation in a hyperpolarizing direction. After membrane depolarization, isoflurane accelerated entry (τ(control)=0.36 [0.03] ms compared with τ(isoflurane)=0.33 [0.05] ms, P<0.05) and slowed recovery (τ(control)=6.9 [1.1] ms compared with τ(isoflurane)=9.0 [1.9] ms, P<0.005) from apparent fast inactivation, resulting in enhanced depression of I(Na), during high-frequency stimulation of both recombinant and endogenous nerve terminal Na(v). A simple model of Na(v) gating involving stabilisation of fast inactivation, accounts for this novel form of activity-dependent block. Isoflurane stabilises the fast-inactivated state of neuronal Na(v) leading to greater depression of I(Na) during high-frequency stimulation, consistent with enhanced inhibition of fast firing neurones.

Frequency-Dependent Inhibition of Sodium Channels by the General Anesthetic Isoflurane

Voltage-gated sodium channels (Nav) are important for initiation and propagation of the action potential in excitable cells, thus contributing to cell-to-cell communication via neurotransmitter release at the synapse. Nav are potential presynaptic targets for inhaled volatile anaesthetics (VAs), which despite their widespread use have poorly understood mechanisms of action. Many drugs that act on Nav, including local anesthetics, show state-dependent effects, that is affinity of the drug depends on whether the channel is open, closed or inactivated. Upon action potential firing, Nav open and rapidly transition into a non-conducting, inactivated state. During high-frequency firing, inactivated channels accumulate as there is insufficient time to return to a closed state before subsequent action potential firing, resulting in frequency-dependent reduction of INa. Therefore, drugs that affect the rates of channel state transitions can have a profound effect on neurotransmitter release and neuronal communication. We investigated the state-dependent effects of the prototypical VA isoflurane on recombinant and native Nav during high-frequency firing. We expressed Nav1.2a, a major isoform found in the central nervous system, in the neuronal mammalian cell line ND7/23 and found that isoflurane enhanced frequency-dependent reduction of INa by enhancing apparent inactivation. To confirm these results in a more physiological preparation we studied the effects of isoflurane on native Nav in rat neurohypophysial nerve terminals and show similar frequency-dependent findings. These data led us to propose a simple pharmacological model of open channel block to account for the effects of isoflurane on Nav1.2. Our data indicate that high-frequency neuronal firing potentiates isoflurane effects, which might contribute to selective modulation of fast firing neuronal networks.Supported by NIH grant GM 58055.

Ca 2+ homeostasis, Ca 2+ signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones

Multiple mechanisms that maintain Ca 2+ homeostasis and provide for Ca 2+ signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca 2+ clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca 2+ homeostatic molecules on cytosolic Ca 2+ ([Ca 2+] i) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca 2+] i dynamics. When bathing the cells in a Na +-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca 2+-ATPase (PMCA), La 3+, all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca 2+] i transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca 2+ transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca 2+ homeostatic pathways, the Na +/Ca 2+ exchanger, the endoplasmic reticulum Ca 2+ pump, the plasmalemmal Ca 2+ pump and mitochondria, are complementary in actively clearing Ca 2+ from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca 2+] i dynamics; (iii) there is (are) Ca 2+ clearance mechanism(s) distinct from the four outlined above; and (iv) Ca 2+ homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.

Blockade of phosphodiesterase Type 5 enhances rat neurohypophysial excitability and electrically evoked oxytocin release.

Phosphodiesterase type 5 (PDE5) acts specifically on cyclic guanosine monophosphate (cGMP) and terminates cGMP-mediated signalling. PDE5 has a well established role in vascular smooth muscle, where specific inhibitors of PDE5 such as sildenafil correct erectile dysfunction by augmenting cGMP-mediated vascular relaxation. However, the role of PDE5 outside of the vasculature has received little attention. The present study tested PDE5 inhibitors on the cGMP-mediated modulation of K(+) channels in the neurohypophysis (posterior pituitary). Photolysis of caged-cGMP enhanced current through Ca(2+)-activated K(+) channels, and this enhancement recovered in about 2 min. Sildenafil essentially eliminated this recovery, suggesting that the reversal of K(+) current enhancement depends on cGMP breakdown. Activation of nitric oxide synthase during trains of activity in pituitary nerve terminals enhances excitability. When trains of stimulation were applied at regular intervals, sildenafil enhanced the excitability of neurohypophysial nerve terminals and increased the action potential firing probability. T-1032, a compound with high specificity for PDE5 over PDE6, had a similar action. Voltage imaging in intact neurohypophysis with a voltage sensitive absorbance dye showed that T-1032 reduced the failure of propagating action potentials during trains of activity. This indicates that PDE5 activity limits action potential propagation in neurohypophysial axons. Immunoassay of oxytocin, a neuropeptide hormone secreted by the posterior pituitary, demonstrated that sildenafil increased electrically evoked release. Thus, PDE5 plays an important role in the regulation of neurohypophysial function, and blockade of this enzyme can enhance the use-dependent facilitation of neurohypophysial secretion.

Depression by isoflurane of the action potential and underlying voltage-gated ion currents in isolated rat neurohypophysial nerve terminals.

We characterized the effects of the volatile anesthetic isoflurane on the ion currents that contribute to the action potential (AP) in isolated rat neurohypophysial (NHP) nerve terminals using patch-clamp electrophysiology. Mean resting membrane potential and AP amplitude were -62.3 +/- 4.1 and 69.2 +/- 2.9 mV, respectively, in NHP terminals. Two components of outward K(+) current (I(K)) were identified in voltage-clamp recordings: a transient I(K) and a sustained I(K) with minimal inactivation. Some terminals displayed a slowly activating I(K), probably the big Ca(2+)-activated K(+) current (BK). Isoflurane reversibly inhibited AP amplitude and increased AP half-width in normal extracellular Ca(2+) (2.2 mM). In high extracellular Ca(2+) (10 mM), isoflurane also reduced the afterhypolarization peak amplitude. A transient tetrodotoxin-sensitive Na(+) current (I(Na)) was the principal current mediating the depolarizing phase of the AP. A slowly inactivating Cd(2+)-sensitive current (probably a voltagegated Ca(2+) current; I(Ca)) followed the initial I(Na). Isoflurane reversibly inhibited both I(Na) and I(Ca) elicited by a voltage-stimulus based on an averaged AP waveform. The isoflurane IC(50) for AP waveform-evoked I(Na) was 0.36 mM. Isoflurane (0.84 +/- 0.04 mM) inhibited AP waveform-evoked I(Ca) by 37.5 +/- 0.16% (p < 0.05). The isoflurane IC(50) for peak I(K) was 0.83 mM and for sustained I(K) was 0.73 mM, with no effect on the voltage dependence of activation. The results indicate that multiple voltage-gated ion channels (Na(+) > K(+) > Ca(2+)) in NHP terminals, although not typical central nervous system terminals, are inhibited by the volatile general anesthetic isoflurane. The net inhibitory effects of volatile anesthetics on nerve terminal action potentials and excitability result from integrated actions on multiple voltage-gated currents.

Ca(2+) and frequency dependence of exocytosis in isolated somata of magnocellular supraoptic neurones of the rat hypothalamus.

In addition to action potential-evoked exocytotic release at neurohypophysial nerve terminals, the neurohormones arginine vasopressin (aVP) and oxytocin (OT) undergo Ca(2+)-dependent somatodendritic release within the supraoptic and paraventricular hypothalamic nuclei. However, the cellular and molecular mechanisms that underlie this release have not been elucidated. In the present study, the whole-cell patch-clamp technique was utilized in combination with high-time-resolved measurements of membrane capacitance (C(m)) and microfluorometric measurements of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) to examine the Ca(2+) and stimulus dependence of exocytosis in the somata of magnocellular neurosecretory cells (MNCs) isolated from rat supraoptic nucleus (SON). Single depolarizing steps (> or =20 ms) that evoked high-voltage-activated (HVA) Ca(2+) currents (I(Ca)) and elevations in intracellular Ca(2+) concentration were accompanied by an increase in C(m) in a majority (40/47) of SON neurones. The C(m) responses were composed of an initial Ca(2+)-independent, transient component and a subsequent, sustained phase of increased C(m) (termed DeltaC(m)) mediated by an influx of Ca(2+), and increased with corresponding prolongation of depolarizing step durations (20-200 ms). From this relationship we estimated the rate of vesicular release to be 1533 vesicles s(-1). Delivery of neurone-derived action potential waveforms (APWs) as stimulus templates elicited I(Ca) and also induced a DeltaC(m), provided APWs were applied in trains of greater than 13 Hz. A train of APWs modelled after the bursting pattern recorded from an OT-containing neurone during the milk ejection reflex was effective in supporting an exocytotic DeltaC(m) in isolated MNCs, indicating that the somata of SON neurones respond to physiological patterns of neuronal activity with Ca(2+)-dependent exocytotic activity.

Impaired somatodendritic responses to pituitary adenylate cyclase-activating polypeptide (PACAP) of supraoptic neurones in PACAP type I -receptor deficient mice.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor (PAC1 receptor) in regulating hypothalamic supraoptic neurones was investigated using PAC1 receptor-deficient male mice (PAC1-/-). The effects of PACAP on [Ca2+]i were investigated in freshly dissociated supraoptic neurones and on the somatodendritic release of vasopressin and oxytocin, examined on intact supraoptic nuclei. In supraoptic neurones from wild-type mice (PAC1+/+), 100 nm PACAP induced an increase in [Ca2+]i and release of vasopressin and oxytocin, whereas in heterozygous (PAC1+/-) and null-mutant mice (PAC1-/-), PACAP was much less effective. PACAP had no effect on these two parameters when applied to isolated neurohypophysial nerve terminals of PAC1+/+ and PAC1-/- mice, and rats. In conclusion, the PAC1 receptor is solely responsible for the PACAP-induced [Ca2+]i signalling and secretion of vasopressin and oxytocin in the somatodendritic region of supraoptic neurones.

Isoflurane and propofol inhibit voltage-gated sodium channels in isolated rat neurohypophysial nerve terminals.

Mounting electrophysiological evidence indicates that certain general anesthetics, volatile anesthetics in particular, depress excitatory synaptic transmission by presynaptic mechanisms. We studied the effects of representative general anesthetics on voltage-gated Na+ currents (INa) in nerve terminals isolated from rat neurohypophysis using patch-clamp electrophysiological analysis. Both isoflurane and propofol inhibited INa in a dose-dependent and reversible manner. At holding potentials of -70 or -90 mV, isoflurane inhibited peak INa with IC50 values of 0.45 and 0.56 mM, and propofol inhibited peak INa with IC50 values of 4.1 and 6.0 microM, respectively. Isoflurane (0.8 mM) did not significantly alter the V1/2 of activation; propofol caused a small positive shift. Isoflurane (0.8 mM) or propofol (5 microM) produced a negative shift in the voltage dependence of inactivation. Recovery of INa from inactivation was slower from a holding potential of -70 mV than from -90 mV; isoflurane and propofol further delayed recovery from inactivation. In conclusion, the volatile anesthetic isoflurane and the intravenous anesthetic propofol inhibit voltage-gated Na+ currents in isolated neurohypophysial nerve terminals in a concentration- and voltage-dependent manner. Marked effects on the voltage dependence and kinetics of inactivation and minimal effects on activation support preferential anesthetic interactions with the fast inactivated state of the Na+ channel. These results are consistent with direct inhibition of oxytocin and vasopressin release from the neurohypophysis by isoflurane and propofol. Inhibition of voltage-gated Na+ channels may contribute to the presynaptic effects of general anesthetics on nerve terminal excitability and neurotransmitter release.

Targeting of green fluorescent protein to secretory granules in oxytocin magnocellular neurons and its secretion from neurohypophysial nerve terminals in transgenic mice.

Oxytocin (OT) is a hypothalamic nonapeptide that is synthesized as part of a larger precursor protein that also contains an approximately 10-kDa protein called neurophysin at its C-terminus. This precursor protein is trafficked through the regulated secretory pathway into secretory granules and then axonally transported to and secreted from nerve terminals in the neural lobe of the pituitary. In this paper, we show that the AI-03 transgene that contains enhanced green fluorescent protein (EGFP) fused to the end of the neurophysin at the C-terminus of the OT pre-prohormone, is expressed selectively in OT-magnocellular neurons and is trafficked to secretory granules in transgenic mice. The EGFP-containing secretory granules are then transported to OT-neurosecretory terminals in the neurohypophysis, where the EGFP fluorescence undergoes depolarization-induced calcium-dependent secretion. The endogenous fluorescence in the neural lobes is sufficiently intense to image secretory events in individual OT nerve terminals (neurosecretosomes) isolated from the posterior pituitaries in these transgenic mice.

Targeting of Green Fluorescent Protein to Secretory Granules in Oxytocin Magnocellular Neurons and Its Secretion from Neurohypophysial Nerve Terminals in Transgenic Mice

Oxytocin (OT) is a hypothalamic nonapeptide that is synthesized as part of a larger precursor protein that also contains an approximately 10-kDa protein called neurophysin at its C-terminus. This precursor protein is trafficked through the regulated secretory pathway into secretory granules and then axonally transported to and secreted from nerve terminals in the neural lobe of the pituitary. In this paper, we show that the AI-03 transgene that contains enhanced green fluorescent protein (EGFP) fused to the end of the neurophysin at the C-terminus of the OT pre-prohormone, is expressed selectively in OT-magnocellular neurons and is trafficked to secretory granules in transgenic mice. The EGFP-containing secretory granules are then transported to OT-neurosecretory terminals in the neurohypophysis, where the EGFP fluorescence undergoes depolarization-induced calcium-dependent secretion. The endogenous fluorescence in the neural lobes is sufficiently intense to image secretory events in individual OT nerve terminals (neurosecretosomes) isolated from the posterior pituitaries in these transgenic mice.

Evidence for endogenous agmatine in hypothalamo-neurohypophysial tract and its modulation on vasopressin release and Ca 2+ channels

Agmatine, decarboxylated from arginine by arginine decarboxylase, is particularly prominent in the hypothalamus. The present study utilized the rat hypothalamo-neurohypophysial system to determine expression and ‘pre-synaptic’ modulation of agmatine in the central nervous system (CNS). Under confocal-laser scanning, agmatine-like immunoreactivity (Agm-LI) was found enriched in arginine–vasopressin (AVP)-containing magnocellular neurons of the supraoptic nuclei (SON) and paraventricular nuclei (PVN). In addition, using electron microscopy, Agm-LI was found closely associated with large neurosecretory-like vesicles in neurohypophysial nerve terminals of posterior pituitary gland. Radioimmunoassay revealed that 10 and 30 μM agmatine concentration-dependently inhibited the depolarization-evoked AVP release from isolated neurohypophysial terminals. Using perforated patch-clamp, effects of agmatine on whole-terminal voltage-gated ion currents in the isolated neurohypophysial nerve terminals were examined. While it did not significantly affect either tetrodotoxin (TTX)-sensitive Na + or sustained Ca 2+-activated K + channel currents, agmatine (1–40 μM) inhibited Ca 2+ channel currents in approximately 53% of the total nerve terminals investigated. The onset of inhibitory effect was immediate, and the inhibition was reversible and concentration-dependent with an IC 50=4.6 μM. In the remaining (approximately 47%) neurohypophysial nerve terminals, only a higher (120 μM) concentration of agmatine could moderately inhibit Ca 2+ channel currents. The results suggest that: (1) endogenous agmatine is co-expressed in AVP-containing, hypothalamic magnocellular neurons of the SON/PVN and in neurohypophysial nerve terminals of posterior pituitary gland; (2) agmatine may serve as a physiological neuromodulator by regulating the voltage-gated Ca 2+ channel and, as a result, the release of AVP from neurohypophysial nerve terminals.

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.

ATP induces intracellular calcium increases and actin cytoskeleton disaggregation via P2x receptors

The consequences of purinoceptor activation on calcium signalling, inositol phosphate metabolism, protein secretion and the actin cytoskeleton were demonstrated in the WRK-1 cell line. Extracellular ATP was used as a secretagogue to induce a rise in int racellular Ca2+concentration ([Ca2+]i), acting via P2x purinergic receptors, which causes actin skeleton disaggregation and protein secretion. ATP bound specifically to purinergic receptors, with Ki of 0.8 μM. The magnitude order for binding of different nucleotides was α β-Met-ATP ≥ dATPαS > ATP ≥ ADP > UTP > AMP > suramin. No increase in inositol phosphates (IPs) was observed after ATP application suggesting that the purinergic sites in WRK-1 cells are not of a P2y type. ATP (1–100 μM) caused a concentration-dependent increase in [Ca2+]i(EC50= 30 μM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca2+]e) was reduced to 100 nM. A non-specific purinergic antagonist, suramin, reversibly inhibited the ATP-response suggesting that ATP is able to bind to P2x purinergic sites to trigger Ca2+entry and increase of [Ca2+]i. ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca2+]e. Similarly, α,β-Met-ATP, a potent P2x agonist also stimulated Ca2+mobilization, actin network destructuration, and protein release. In the isolated rat neurohypophysial nerve terminals, ATP was shown to act as a physiological stimulus for vasopressin release via Ca2+entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is also able to induce actin disaggregation by a Ca2+dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis.

Agmatine Is an Autocrine/Paracrine Inhibitor of Pre-Synaptic Ca ++ Channels and Vasopressin Release in Vasopressinergic Neurons of the Hypothalamo-Hypophyseal Tract

20 Agmatine (decarboxylated arginine), an organic cation, endogenous ligand at imidazoline (I-) and α 2 -adrenergic receptors, antagonist of NMDA receptors and inhibitor of nitric oxide synthase, may be a novel neurotransmitter in mammalian CNS (Reis and Regunathan, TIPS, 2000). To further establish a cellular function of agmatine we investigated whether it is expressed in, and regulates the function of, magnocellular neurons of the hypothalamic paraventricular (PVH)/supraoptic (SON) nuclei in rat. By LM, agmatine-like immunoreactivity (Ag-LI) was present in virtually all magnocellular PVH and SON neurons, where it co-localized with arginine vasopressin (AVP) and oxytocin (OXY). By EM, Ag-LI was present in large dense core vesicles in neurohypophysial nerve terminals (NNTs), known storage sites of the peptides. NNTs acutely isolated from adult rats were dispersed and voltage-activated Na + , K + , and Ca ++ currents recorded in whole terminals in amphotericin B perforated-patches. Agmatine (40-120 uM) did not affect the voltage-dependent, TTX-sensitive inward Na + , the 4-AP-sensitive, transient outward A-type K + current, or the sustained, Ca ++ -activated, maxi-conductance K + currents. In ∼ 60% of terminals 40 uM agmatine immediately, dose-dependently and reversibly blocked whole cell Ca ++ currents evoked by 10 mM Ca ++ with an IC 50 ∼ 5 uM. Agmatine blocked the depolarization-induced release of AVP from isolated NNTs. We conclude that agmatine: (a) is co-stored with neuropeptides in hypothalamo-neurohypophysial neurons, and (b) inhibits pre-synaptic Ca ++ channels to (c) inhibit release of AVP. Agmatine is a neurotransmitter in AVP neurons of PVH and SON, wherein it functions as a paracrine/autocrine inhibitor of AVP release by blocking Ca ++ channels. Agmatine may regulate neurally integrated release of this important hormone, regulating its cardiovascular and renal functions.

Agmatine Is an Autocrine/Paracrine Inhibitor of Pre-Synaptic Ca++ Channels and Vasopressin Release in Vasopressinergic Neurons of the Hypothalamo-Hypophyseal Tract

20 Agmatine (decarboxylated arginine), an organic cation, endogenous ligand at imidazoline (I-) and α 2 -adrenergic receptors, antagonist of NMDA receptors and inhibitor of nitric oxide synthase, may be a novel neurotransmitter in mammalian CNS (Reis and Regunathan, TIPS, 2000). To further establish a cellular function of agmatine we investigated whether it is expressed in, and regulates the function of, magnocellular neurons of the hypothalamic paraventricular (PVH)/supraoptic (SON) nuclei in rat. By LM, agmatine-like immunoreactivity (Ag-LI) was present in virtually all magnocellular PVH and SON neurons, where it co-localized with arginine vasopressin (AVP) and oxytocin (OXY). By EM, Ag-LI was present in large dense core vesicles in neurohypophysial nerve terminals (NNTs), known storage sites of the peptides. NNTs acutely isolated from adult rats were dispersed and voltage-activated Na + , K + , and Ca ++ currents recorded in whole terminals in amphotericin B perforated-patches. Agmatine (40-120 uM) did not affect the voltage-dependent, TTX-sensitive inward Na + , the 4-AP-sensitive, transient outward A-type K + current, or the sustained, Ca ++ -activated, maxi-conductance K + currents. In ∼ 60% of terminals 40 uM agmatine immediately, dose-dependently and reversibly blocked whole cell Ca ++ currents evoked by 10 mM Ca ++ with an IC 50 ∼ 5 uM. Agmatine blocked the depolarization-induced release of AVP from isolated NNTs. We conclude that agmatine: (a) is co-stored with neuropeptides in hypothalamo-neurohypophysial neurons, and (b) inhibits pre-synaptic Ca ++ channels to (c) inhibit release of AVP. Agmatine is a neurotransmitter in AVP neurons of PVH and SON, wherein it functions as a paracrine/autocrine inhibitor of AVP release by blocking Ca ++ channels. Agmatine may regulate neurally integrated release of this important hormone, regulating its cardiovascular and renal functions.

Membrane-delimited coupling between sigma receptors and K+ channels in rat neurohypophysial terminals requires neither G-protein nor ATP.

Receptor-mediated modulation of ion channels generally involves G-proteins, phosphorylation, or both in combination. The sigma receptor, which modulates voltage-gated K+ channels, is a novel protein with no homology to other receptors known to modulate ion channels. In the present study patch clamp and photolabelling techniques were used to investigate the mechanism by which sigma receptors modulate K+ channels in peptidergic nerve terminals. The sigma receptor photoprobe iodoazidococaine labelled a protein with the same molecular mass (26 kDa) as the sigma receptor protein identified by cloning. The sigma receptor ligands pentazocine and SKF10047 modulated K+ channels, despite intra-terminal perfusion with GTP-free solutions, a G-protein inhibitor (GDPbetaS), a G-protein activator (GTPgammaS) or a non-hydrolysable ATP analogue (AMPPcP). Channels in excised outside-out patches were modulated by ligand, indicating that soluble cytoplasmic factors are not required. In contrast, channels within cell-attached patches were not modulated by ligand outside a patch, indicating that receptors and channels must be in close proximity for functional interactions. Channels expressed in oocytes without receptors were unresponsive to sigma receptor agonists, ruling out inhibition through a direct drug interaction with channels. These experiments indicate that sigma receptor-mediated signal transduction is membrane delimited, and requires neither G-protein activation nor protein phosphorylation. This novel transduction mechanism is mediated by membrane proteins in close proximity, possibly through direct interactions between the receptor and channel. This would allow for more rapid signal transduction than other ion channel modulation mechanisms, which in the present case of neurohypophysial nerve terminals would lead to the enhancement of neuropeptide release.

K+ channel modulation in rodent neurohypophysial nerve terminals by sigma receptors and not by dopamine receptors.

1. Sigma receptors bind a diverse group of chemically unrelated ligands, including pentazocine, apomorphine (a dopamine receptor agonist) and haloperidol (a dopamine receptor antagonist). Although sigma binding sites are widely distributed, their physiological roles are poorly understood. Here, the whole-terminal patch-clamp technique was used to demonstrate that sigma receptors modulate K+ channels in rodent neurohypophysis. 2. Previous work suggested that dopamine type 4 (D4) receptors modulate neurohypophysial K+ current, so this study initially tested the role of dopamine receptors. Experiments using transgenic mice lacking D2, D3 or D4 receptors indicated that the reduction of K+ current by PPHT and U101958 (ligands thought to be selective for dopamine receptors) is not mediated by dopamine receptors. The sensitivity of the response to U101958 (a drug that binds to D4 receptors) was the same in both wild-type and D4 receptor-deficient mice. 3. Experiments with other ligands revealed a pharmacological signature inconsistent with any known dopamine receptor. Furthermore, dopamine itself (at 100 microM) had no effect. Thus, despite the activity of a number of putative dopamine receptor ligands, dopamine receptors play no role in the modulation of neurohypophysial K+ channels. 4. Because of the negative results regarding dopamine receptors, and because some of the dopamine receptors ligands used here are known to bind also to sigma receptors, experiments were conducted to test for the involvement of sigma receptors. In rat neurohypophysis the sigma receptor ligands SKF10047, pentazocine, and ditolylguanidine all reversibly inhibited K+ current in a concentration-dependent fashion, as did haloperidol and apomorphine (ligands that bind to both dopamine and sigma receptors). The activity of these and other ligands tested here matches the reported binding specificity for sigma receptors. 5. Fifteen candidate endogenous sigma receptor ligands, including biogenic amines (e.g dopamine and serotonin), steroids (e.g. progesterone), and peptides (e.g. neuropeptide Y), were screened for activity at the sigma receptor. All were without effect. 6. Haloperidol reduced K+ current proportionally at all voltages without shifting the voltage dependence of activation and inactivation. Sigma receptor ligands inhibited current through two distinct K+ channels, the A-channel and the Ca2+-dependent K+ channel. In rat, all drugs reduced current through both channels proportionally, suggesting that both channels are modulated by a single population of sigma receptors. In contrast, mouse peptidergic nerve terminals either have two receptors which are sensitive to these drugs, or a single receptor that is differentially coupled to ion channel function. 7. The inhibition of voltage-activated K+ current by sigma receptors would be expected to enhance the secretion of oxytocin and vasopressin from the neurohypophysis.

ATP-evoked increases in [Ca2+]i and peptide release from rat isolated neurohypophysial terminals via a P2X2 purinoceptor.

1. The effect of externally applied ATP on cytosolic free Ca2+ concentration ([Ca2+]i) was tested in single isolated rat neurohypophysial nerve terminals by fura-2 imaging. The release of vasopressin (AVP) and oxytocin (OT) upon ATP stimulation was also studied from a population of terminals using specific radioimmunoassays. 2. ATP evoked a sustained [Ca2+]i increase, which was dose dependent in the 1-100 microM range (EC50 = 4.8 microM). This effect was observed in only approximately 40 % of the terminals. 3. Interestingly, ATP, in the same range (EC50 = 8.6 microM), evoked AVP, but no significant OT, release from these terminals. 4. Both the [Ca2+]i increase and AVP release induced by ATP were highly and reversibly inhibited by suramin, suggesting the involvement of a P2 purinergic receptor in the ATP-induced responses. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), another P2 purinergic receptor antagonist, strongly reduced the ATP-induced [Ca2+]i response. 5. To further characterize the receptor, different agonists were tested, with the following efficacy: ATP = 2-methylthio-ATP > ATP-gamma-S > alpha, beta-methylene-ATP > ADP. The compounds adenosine, AMP, beta, gamma-methylene-ATP and UTP were ineffective. 6. The ATP-dependent [Ca2+]i increase was dependent on extracellular Ca2+ concentration ([Ca2+]o). Fluorescence-quenching experiments with Mn2+ showed that externally applied ATP triggered a Mn2+ influx. The ATP-induced [Ca2+]i increase and AVP release were independent of and additive to a K+-induced response, in addition to being insensitive to Cd2+. The ATP-induced [Ca2+]i increase was strongly reduced in the presence of Gd3+. These results suggest that the observed [Ca2+]i increases were elicited by Ca2+ entry through a P2X channel receptor rather than via a voltage-dependent Ca2+ channel. 7. We propose that ATP, co-released with neuropeptides, could act as a paracrine-autocrine messenger, stimulating, via Ca2+ entry through a P2X2 receptor, the secretion of AVP, in particular, from neurohypophysial nerve terminals.

Calcium-induced calcium increase in secretory vesicles of permeabilized rat neurohypophysial nerve terminals

Digitonin-permeabilized isolated neurohypophysial nerve terminals are known to release their secretory vesicle content under calcium challenge. On this preparation, we monitored intra-organelle Ca 2+ concentration using digital fluorescence microscopy of Fura-2. The superfusion of artificial intracellular solution containing 10 to 50 μM Ca 2+ induced an intra-organelle [Ca 2+] increase. Two major organelles are candidates for this increase: secretory vesicles and mitochondria. In an attempt to detect calcium changes in the vesicles, ruthenium red was used to impair mitochondrial calcium uptake. Part of the ruthenium red-insensitive intra-organelle [Ca 2+] increase was abolished by raising sodium in the solution. Removing sodium boosted the intra-organelle [Ca 2+] increase. These results taken together suggest the participation of Na Ca exchange, known to exist in the membrane of these secretory vesicles. In addition to Na Ca exchange, there would be at least another mechanism of vesicular calcium intake, as suggested by the partial inhibition of intra-organelle [Ca 2+] increase obtained under acidic compartments: neutralization with NH 4Cl. This mechanism remains to be defined. The main conclusion presented here, that an intravesicular [Ca 2+] increase takes place at the rate of secretion, was predicted by the hypothesis that intravesicular Ca 2+ changes would be involved in stimulus-secretion coupling.

Calcium‐induced calcium increase in secretory vesicles of permeabilized rat neurohypophysial nerve terminals

Digitonin-permeabilized isolated neurohypophysial nerve terminals are known to release their secretory vesicle content under calcium challenge. On this preparation, we monitored intra-organelle Ca2+ concentration using digital fluorescence microscopy of Fura-2. The superfusion of artificial intracellular solution containing 10 to 50 microM Ca2+ induced an intra-organelle [Ca2+] increase. Two major organelles are candidates for this increase: secretory vesicles and mitochondria. In an attempt to detect calcium changes in the vesicles, ruthenium red was used to impair mitochondrial calcium uptake. Part of the ruthenium red-insensitive intra-organelle [Ca2+] increase was abolished by raising sodium in the solution. Removing sodium boosted the intra-organelle [Ca2+] increase. These results taken together suggest the participation of Na/Ca exchange, known to exist in the membrane of these secretory vesicles. In addition to Na/Ca exchange, there would be at least another mechanism of vesicular calcium intake, as suggested by the partial inhibition of intra-organelle [Ca2+] increase obtained under acidic compartments: neutralization with NH4Cl. This mechanism remains to be defined. The main conclusion presented here, that an intravesicular [Ca2+] increase takes place at the rate of secretion, was predicted by the hypothesis that intravesicular Ca2+ changes would be involved in stimulus-secretion coupling.