µ-Opioid inhibition of Ca2+ currents and secretion in isolated terminals of the neurohypophysis occurs via ryanodine-sensitive Ca2+ stores.

µ-Opioid agonists have no effect on calcium currents (I(Ca)) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, µ-opioid receptor (MOR)-mediated inhibition of I(Ca) is reliably demonstrated using the perforated-patch configuration. This suggests that the MOR-signaling pathway is sensitive to intraterminal dialysis and is therefore mediated by a readily diffusible second messenger. Using the perforated patch-clamp technique and ratio-calcium-imaging methods, we describe a diffusible second messenger pathway stimulated by the MOR that inhibits voltage-gated calcium channels in isolated terminals from the rat neurohypophysis (NH). Our results show a rise in basal intracellular calcium ([Ca(2+)]i) in response to application of [D-Ala(2)-N-Me-Phe(4),Gly5-ol]-Enkephalin (DAMGO), a MOR agonist, that is blocked by D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a MOR antagonist. Buffering DAMGO-induced changes in [Ca(2+)]i with BAPTA-AM completely blocked the inhibition of both I(Ca) and high-K(+)-induced rises in [Ca(2+)]i due to MOR activation, but had no effect on κ-opioid receptor (KOR)-mediated inhibition. Given the presence of ryanodine-sensitive stores in isolated terminals, we tested 8-bromo-cyclic adenosine diphosphate ribose (8Br-cADPr), a competitive inhibitor of cyclic ADP-ribose (cADPr) signaling that partially relieves DAMGO inhibition of I(Ca) and completely relieves MOR-mediated inhibition of high-K(+)-induced and DAMGO-induced rises in [Ca(2+)]i. Furthermore, antagonist concentrations of ryanodine completely blocked MOR-induced increases in [Ca(2+)]i and inhibition of I(Ca) and high-K(+)-induced rises in [Ca(2+)]i while not affecting KOR-mediated inhibition. Antagonist concentrations of ryanodine also blocked MOR-mediated inhibition of electrically-evoked increases in capacitance. These results strongly suggest that a key diffusible second messenger mediating the MOR-signaling pathway in NH terminals is [Ca(2+)]i released by cADPr from ryanodine-sensitive stores.

P2X7 receptors in neurohypophysial terminals: evidence for their role in arginine-vasopressin secretion.

Arginine-vasopressin (AVP) plays a major role in maintaining cardiovascular function and related pathologies. The mechanism involved in its release into the circulation is complex and highly regulated. Recent work has implicated the purinergic receptor, P2X7R, in a role for catecholamine-enhanced AVP release in the rat hypothalamic-neurohypophysial (NH) system. However, the site of P2X7R action in this endocrine system, and whether or not it directly mediates release in secretory neurons have not been determined. We hypothesized that the P2X7R is expressed and mediates AVP release in NH terminals. P2X7R function was first examined by patch-clamp recordings in isolated NH terminals. Results revealed that subpopulations of isolated terminals displayed either high ATP-sensitivity or low ATP-sensitivity, the latter of which was characteristic of the rat P2X7R. Additional recordings showed that terminals showing sensitivity to the P2X7R-selective agonist, BzATP, were further inhibited by P2X7R selective antagonists, AZ10606120 and brilliant blue-G. In confocal micrographs from tissue sections and isolated terminals of the NH P2X7R-immunoreactivity was found to be localized in plasma membranes. Lastly, the role of P2X7R on AVP release was tested. Our results showed that BzATP evoked sustained AVP release in NH terminals, which was inhibited by AZ10606120. Taken together, our data lead us to conclude that the P2X7R is expressed in NH terminals and corroborates its role in AVP secretion.

Differential modulation of N-type calcium channels by micro-opioid receptors in oxytocinergic versus vasopressinergic neurohypophysial terminals.

Opioids modulate the electrical activity of magnocellular neurons (MCN) and inhibit neuropeptide release at their terminals in the neurohypophysis. We have previously shown that micro-opioid receptor (MOR) activation induces a stronger inhibition of oxytocin (OT) than vasopressin (AVP) release from isolated MCN terminals. This higher sensitivity of OT release is due, at least in part, to the selective targeting of R-type calcium channels. We now describe the underlying basis for AVP's weaker inhibition by MOR activation and provide a more complete explanation of the complicated effects on neuropeptide release. We found that N-type calcium channels in AVP terminals are differentially modulated by MOR; enhanced at lower concentrations but increasingly inhibited at higher concentrations of agonists. On the other hand, N-type calcium channels in OT terminals were always inhibited. The response pattern in co-labeled terminals was analogous to that observed in AVP-containing terminals. Changes in intracellular calcium concentration and neuropeptide release corroborated these results as they showed a similar pattern of enhancement and inhibition in AVP terminals contrasting with solely inhibitory responses in OT terminals to MOR agonists. We established that fast translocation of Ca(2+) channels to the plasma membrane was not mediating current increments and thus, changes in channel kinetic properties are most likely involved. Finally, we reveal a distinct Ca-channel beta-subunit expression between each type of nerve endings that could explain some of the differences in responses to MOR activation. These results help advance our understanding of the complex modulatory mechanisms utilized by MORs in regulating presynaptic neuropeptide release.

Voltage-induced Ca2+ release by ryanodine receptors causes neuropeptide secretion from nerve terminals.

Depolarisation-secretion coupling is assumed to be dependent only on extracellular calcium ([Ca2+ ]o ). Ryanodine receptor (RyR)-sensitive stores in hypothalamic neurohypophysial system (HNS) terminals produce sparks of intracellular calcium ([Ca2+ ]i ) that are voltage-dependent. We hypothesised that voltage-elicited increases in intraterminal calcium are crucial for neuropeptide secretion from presynaptic terminals, whether from influx through voltage-gated calcium channels and/or from such voltage-sensitive ryanodine-mediated calcium stores. Increases in [Ca2+ ]i upon depolarisation in the presence of voltage-gated calcium channel blockers, or in the absence of [Ca2+ ]o , still give rise to neuropeptide secretion from HNS terminals. Even in 0 [Ca2+ ]o , there was nonetheless an increase in capacitance suggesting exocytosis upon depolarisation. This was blocked by antagonist concentrations of ryanodine, as was peptide secretion elicited by high K+ in 0 [Ca2+ ]o . Furthermore, such depolarisations lead to increases in [Ca2+ ]i . Pre-incubation with BAPTA-AM resulted in > 50% inhibition of peptide secretion elicited by high K+ in 0 [Ca2+ ]o . Nifedipine but not nicardipine inhibited both the high K+ response for neuropeptide secretion and intraterminal calcium, suggesting the involvement of CaV1.1 type channels as sensors in voltage-induced calcium release. Importantly, RyR antagonists also modulate neuropeptide release under normal physiological conditions. In conclusion, our results indicate that depolarisation-induced neuropeptide secretion is present in the absence of external calcium, and calcium release from ryanodine-sensitive internal stores is a significant physiological contributor to neuropeptide secretion from HNS terminals.

Endogenous ATP potentiates only vasopressin secretion from neurohypophysial terminals.

Exogenous ATP induces inward currents and causes the release of arginine-vasopressin (AVP) from isolated neurohypophysial terminals (NHT); both effects are inhibited by the P2X2 and P2X3 antagonists, suramin and PPADS. Here we examined the role of endogenous ATP in the neurohypophysis. Stimulation of NHT caused the release of both AVP and ATP. ATP induced a potentiation in the stimulated release of AVP, but not of oxytocin (OT), which was blocked by the presence of suramin. In loose-patch clamp recordings, from intact neurohypophyses, suramin or PPADS produces an inhibition of action potential currents in a static bath, that can be mimicked by a hyperpolarization of the resting membrane potential (RMP). Correspondingly, in a static versus perfused bath there is a depolarization of the RMP of NHT, which was reduced by either suramin or PPADS. We measured an accumulation of ATP (3.7 +/- 0.7 microM) released from NHT in a static bath. Applications of either suramin or PPADS to a static bath decreased burst-stimulated capacitance increases in NHT. Finally, only vasopressin release from electrically stimulated intact neurohypophyses was reduced in the presence of Suramin or PPADS. These data suggest that there was sufficient accumulation of ATP released from the neurohypophysis during stimulations to depolarize its nerve terminals. This would occur via the opening of P2X2 and P2X3 receptors, inducing an influx of Ca2+. The subsequent elevation in [Ca2+](i) would further increase the stimulated release of only vasopressin from NHT terminals. Such purinergic feedback mechanisms could be physiologically important at most CNS synapses.

Purinergic receptor types in the hypothalamic-neurohypophysial system.

Many different types of purinergic receptors are present in the Hypothalamic-Neurohypophysial System (HNS), which synthesizes and releases vasopressin and oxytocin. The specific location of purinergic receptor subtypes has important functional repercussions for neuronal activity and synaptic output. Yet, until the advent of receptor KOs, this had been hindered by the low selectivity of the available pharmacological tools. The HNS offers an excellent opportunity to differentiate the functional properties of these purinergic receptors in cell bodies vs. terminals of the same physiological system. P2X2, P2X3, P2X4 and P2X7 receptors are present in vasopressin terminals while oxytocin terminals exclusively express the P2X7 subtype. The latter is not functional in the cell bodies of the HNS. These purinergic receptor subtypes are permeable to sodium vs. calcium in varying amounts and this could play an important role in the release of vasopressin vs. oxytocin during bursting activity. Endogenous ATP and its metabolite, adenosine, have autocrine and paracrine modulatory effects on the release of these neuropeptides during physiological stimulation. Finally, we hypothesize that during such action potential bursts, ATP potentiates the release of vasopressin but not of oxytocin, and that adenosine, via A1 receptors, inhibits the release of both neuropeptides. This article is protected by copyright. All rights reserved.

Identification of the neuropeptide content of individual rat neurohypophysial terminals.

The objective of this study was to develop a method that could reliably determine the arginine vasopressin (AVP) and/or oxytocin (OT) content of individual rat neurohypophysial terminals (NHT) >or=5 microm in diameter, the size used for electrophysiological recordings. We used a commercially available, highly sensitive enzyme-linked immunoassay (ELISA) kit with a sensitivity of 0.25 pg to AVP and of 1.0pg to OT. The NHT content of AVP (2.21+/-0.10 pg) was greater than OT (1.77+/-0.08 pg) and increased with terminal size. AVP-positive terminals (10.2+/-0.21 microm) were larger in diameter than OT-positive terminals (9.1+/-0.24 microm). Immunocytochemical techniques indicated that a higher percentage (58%) of smaller terminals contained OT, and that a higher percentage (42%) of larger NHTs were colabeled. Similar percentages of AVP-positive terminals were obtained between immunocytochemical (73%) and ELISA (72%) methods when NHTs were assayed for AVP alone, but there was a higher percentage of OT terminals when using immunocytochemistry (43%) compared to ELISA (26%). The percent of AVP-positive (60%) and OT-positive (18%) terminals decreased when NHT were assayed for both AVP and OT. Therefore, the best method to reliably identify AVP-positive NHTs is to assay only for AVP, since this allows the conclusion that AVP-negative terminals contain only OT.

Functional ryanodine receptors in the membranes of neurohypophysial secretory granules.

Highly localized Ca(2+) release events have been characterized in several neuronal preparations. In mouse neurohypophysial terminals (NHTs), such events, called Ca(2+) syntillas, appear to emanate from a ryanodine-sensitive intracellular Ca(2+) pool. Traditional sources of intracellular Ca(2+) appear to be lacking in NHTs. Thus, we have tested the hypothesis that large dense core vesicles (LDCVs), which contain a substantial amount of calcium, represent the source of these syntillas. Here, using fluorescence immunolabeling and immunogold-labeled electron micrographs of NHTs, we show that type 2 ryanodine receptors (RyRs) are localized specifically to LDCVs. Furthermore, a large conductance nonspecific cation channel, which was identified previously in the vesicle membrane and has biophysical properties similar to that of an RyR, is pharmacologically affected in a manner characteristic of an RyR: it is activated in the presence of the RyR agonist ryanodine (at low concentrations) and blocked by the RyR antagonist ruthenium red. Additionally, neuropeptide release experiments show that these same RyR agonists and antagonists modulate Ca(2+)-elicited neuropeptide release from permeabilized NHTs. Furthermore, amperometric recording of spontaneous release events from artificial transmitter-loaded terminals corroborated these ryanodine effects. Collectively, our findings suggest that RyR-dependent syntillas could represent mobilization of Ca(2+) from vesicular stores. Such localized vesicular Ca(2+) release events at the precise location of exocytosis could provide a Ca(2+) amplification mechanism capable of modulating neuropeptide release physiologically.

Modulation/physiology of calcium channel sub-types in neurosecretory terminals.

The hypothalamic-neurohypophysial system (HNS) controls diuresis and parturition through the release of arginine-vasopressin (AVP) and oxytocin (OT). These neuropeptides are chiefly synthesized in hypothalamic magnocellular somata in the supraoptic and paraventricular nuclei and are released into the blood stream from terminals in the neurohypophysis. These HNS neurons develop specific electrical activity (bursts) in response to various physiological stimuli. The release of AVP and OT at the level of neurohypophysis is directly linked not only to their different burst patterns, but is also regulated by the activity of a number of voltage-dependent channels present in the HNS nerve terminals and by feedback modulators. We found that there is a different complement of voltage-gated Ca(2+) channels (VGCC) in the two types of HNS terminals: L, N, and Q in vasopressinergic terminals vs. L, N, and R in oxytocinergic terminals. These channels, however, do not have sufficiently distinct properties to explain the differences in release efficacy of the specific burst patterns. However, feedback by both opioids and ATP specifically modulate different types of VGCC and hence the amount of AVP and/or OT being released. Opioid receptors have been identified in both AVP and OT terminals. In OT terminals, µ-receptor agonists inhibit all VGCC (particularly R-type), whereas, they induce a limited block of L-, and P/Q-type channels, coupled to an unusual potentiation of the N-type Ca(2+) current in the AVP terminals. In contrast, the N-type Ca(2+) current can be inhibited by adenosine via A(1) receptors leading to the decreased release of both AVP and OT. Furthermore, ATP evokes an inactivating Ca(2+)/Na(+)-current in HNS terminals able to potentiate AVP release through the activation of P2X2, P2X3, P2X4 and P2X7 receptors. In OT terminals, however, only the latter receptor type is probably present. We conclude by proposing a model that can explain how purinergic and/or opioid feedback modulation during bursts can mediate differences in the control of neurohypophysial AVP vs. OT release.

Endogenous adenosine inhibits CNS terminal Ca(2+) currents and exocytosis.

Bursts of action potentials (APs) are crucial for the release of neurotransmitters from dense core granules. This has been most definitively shown for neuropeptide release in the hypothalamic neurohypophysial system (HNS). Why such bursts are necessary, however, is not well understood. Thus far, biophysical characterization of channels involved in depolarization-secretion coupling cannot completely explain this phenomenon at HNS terminals, so purinergic feedback mechanisms have been proposed. We have previously shown that ATP, acting via P2X receptors, potentiates release from HNS terminals, but that its metabolite adenosine, via A(1) receptors acting on transient Ca(2+) currents, inhibit neuropeptide secretion. We now show that endogenous adenosine levels are sufficient to cause tonic inhibition of transient Ca(2+) currents and of stimulated exocytosis in HNS terminals. Initial non-detectable adenosine levels in the static bath increased to 2.9 microM after 40 min. These terminals exhibit an inhibition (39%) of their transient inward Ca(2+) current in a static bath when compared to a constant perfusion stream. CPT, an A(1) adenosine receptor antagonist, greatly reduced this tonic inhibition. An ecto-ATPase antagonist, ARL-67156, similarly reduced tonic inhibition, but CPT had no further effect, suggesting that endogenous adenosine is due to breakdown of released ATP. Finally, stimulated capacitance changes were greatly enhanced (600%) by adding CPT to the static bath. Thus, endogenous adenosine functions at terminals in a negative-feedback mechanism and, therefore, could help terminate peptide release by bursts of APs initiated in HNS cell bodies. This could be a general mechanism for controlling transmitter release in these and other CNS terminals.

Adenosine inhibition via A(1) receptor of N-type Ca(2+) current and peptide release from isolated neurohypophysial terminals of the rat.

Effects of adenosine on voltage-gated Ca(2+) channel currents and on arginine vasopressin (AVP) and oxytocin (OT) release from isolated neurohypophysial (NH) terminals of the rat were investigated using perforated-patch clamp recordings and hormone-specific radioimmunoassays. Adenosine, but not adenosine 5'-triphosphate (ATP), dose-dependently and reversibly inhibited the transient component of the whole-terminal Ba(2+) currents, with an IC(50) of 0.875 microM. Adenosine strongly inhibited, in a dose-dependent manner (IC(50) = 2.67 microM), depolarization-triggered AVP and OT release from isolated NH terminals. Adenosine and the N-type Ca(2+) channel blocker omega-conotoxin GVIA, but not other Ca(2+) channel-type antagonists, inhibited the same transient component of the Ba(2+) current. Other components such as the L-, Q- and R-type channels, however, were insensitive to adenosine. Similarly, only adenosine and omega-conotoxin GVIA were able to inhibit the same component of AVP release. A(1) receptor agonists, but not other purinoceptor-type agonists, inhibited the same transient component of the Ba(2+) current as adenosine. Furthermore, the A(1) receptor antagonist 8-cyclopentyltheophylline (CPT), but not the A(2) receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DMPGX), reversed inhibition of this current component by adenosine. The inhibition of AVP and OT release also appeared to be via the A(1) receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A(1) receptors, specifically blocks the terminal N-type Ca(2+) channel thus leading to inhibition of the release of both AVP and OT.