Edward E. Custer

Ca2+ Syntillas, Miniature Ca2+ Release Events in Terminals of Hypothalamic Neurons, Are Increased in Frequency by Depolarization in the Absence of Ca2+ Influx

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of ∼250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of –80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.

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.

µ‐Opioid Receptor Preferentially Inhibits Oxytocin Release from Neurohypophysial Terminals by Blocking R‐type Ca2+ Channels

Oxytocin release from neurophypophysial terminals is particularly sensitive to inhibition by the µ‐opioid receptor agonist, DAMGO. Because the R‐type component of the neurophypophysial terminal Ca2+ current (ICa) mediates exclusively oxytocin release, we hypothesised that µ‐opioids could preferentially inhibit oxytocin release by blocking this channel subtype. Whole‐terminal recordings showed that DAMGO and the R‐type selective blocker SNX‐482 inhibit a similar ICa component. Measurements of [Ca2+]i levels and oxytocin release confirmed that the effects of DAMGO and SNX‐482 are not additive. Finally, isolation of the R‐type component and its associated rise in [Ca2+]i and oxytocin release allowed us to demonstrate the selective inhibition by DAMGO of this channel subtype. Thus, µ‐opioid agonists modulate specifically oxytocin release in neurophypophysial terminals by selectively targeting R‐type Ca2+ channels. Modulation of Ca2+ channel subtypes could be a general mechanism for drugs of abuse to regulate the release of specific neurotransmitters at central nervous system synapses.

Adenosine inhibition via A1 receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat

Effects of adenosine on voltage‐gated Ca2+ 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 Ba2+ currents, with an IC50 of 0.875 μm. Adenosine strongly inhibited, in a dose‐dependent manner (IC50= 2.67 μm), depolarization‐triggered AVP and OT release from isolated NH terminals. Adenosine and the N‐type Ca2+ channel blocker ω‐conotoxin GVIA, but not other Ca2+ channel‐type antagonists, inhibited the same transient component of the Ba2+ current. Other components such as the L‐, Q‐ and R‐type channels, however, were insensitive to adenosine. Similarly, only adenosine and ω‐conotoxin GVIA were able to inhibit the same component of AVP release. A1 receptor agonists, but not other purinoceptor‐type agonists, inhibited the same transient component of the Ba2+ current as adenosine. Furthermore, the A1 receptor antagonist 8‐cyclopentyltheophylline (CPT), but not the A2 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 A1 receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A1 receptors, specifically blocks the terminal N‐type Ca2+ channel thus leading to inhibition of the release of both AVP and OT.

µ‐Opioid Receptor Modulates Peptide Release From Rat Neurohypophysial Terminals By Inhibiting Ca2+ Influx

The activation of opioid receptors in neurones of the central nervous system leads to a variety of effects including the modulation of diuresis and parturition, processes that are directly controlled by the hypothalamic–neurohypophysial system (HNS). The effects of µ‐opioid receptor activation on peptide release, voltage‐gated Ca2+ currents and intracellular calcium levels ([Ca2+]i) were studied in isolated nerve terminals of the HNS. The µ‐receptor agonist, DAMGO ([d‐Ala2,N‐Me‐Phe4,Gly5‐ol]‐enkephalin) inhibited high K+‐induced peptide release in a dose‐dependent manner, with oxytocin release being more sensitive to block than vasopressin release at all concentrations tested. The addition of the µ‐receptor antagonist CTOP (d‐Phe‐Cys‐Tyr‐d‐Trp‐Orn‐Thr‐Pen‐Thr amide) was able to overcome the inhibitory effects of DAMGO. By contrast to previous results, voltage‐gated Ca2+ currents were sensitive to blockage by DAMGO and this inhibition was also prevented by CTOP. Furthermore, [Ca2+]i measurements with Fura‐2 corroborated the inhibition by DAMGO of calcium entry and its reversal by the µ‐receptor antagonist in these nerve terminals. Thus, the decrease in neuropeptide release, particularly for oxytocin, induced by the activation of µ‐opioid receptors in neurohypophysial terminals is mediated, at least in part, by a corresponding decrease in Ca2+ entry due to the inhibition of voltage‐gated Ca2+ channels.

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 µM) 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. J. Cell. Physiol. 217: 155–161, 2008.

Endogenous adenosine inhibits CNS terminal Ca2+ 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 A1 receptors acting on transient Ca2+ currents, inhibit neuropeptide secretion. We now show that endogenous adenosine levels are sufficient to cause tonic inhibition of transient Ca2+ currents and of stimulated exocytosis in HNS terminals. Initial non‐detectable adenosine levels in the static bath increased to 2.9 µM after 40 min. These terminals exhibit an inhibition (39%) of their transient inward Ca2+ current in a static bath when compared to a constant perfusion stream. CPT, an A1 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. J. Cell. Physiol. 210: 309–314, 2007.

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.

P2X purinergic receptor knockout mice reveal endogenous ATP modulation of both vasopressin and oxytocin release from the intact neurohypophysis.

Bursts of action potentials are crucial for neuropeptide release from the hypothalamic neurohypophysial system (HNS). The biophysical properties of the ion channels involved in the release of these neuropeptides, however, cannot explain the efficacy of such bursting patterns on secretion. We have previously shown that ATP, acting via P2X receptors, potentiates only vasopressin (AVP) release from HNS terminals, whereas its metabolite adenosine, via A1 receptors acting on transient Ca2+ currents, inhibits both AVP and oxytocin (OT) secretion. Thus, purinergic feedback‐mechanisms have been proposed to explain bursting efficacy at HNS terminals. Therefore, in the present study, we have used specific P2X receptor knockout (rKO) mice and purportedly selective P2X receptor antagonists to determine the P2X receptor subtype responsible for endogenous ATP induced potentiation of electrically‐stimulated neuropeptide release. Intact neurohypophyses (NH) from wild‐type (WT), P2X3 rKO, P2X2/3 rKO and P2X7 rKO mice were electrically stimulated with four 25‐s bursts (3 V at 39 Hz) separated by 21‐s interburst intervals with or without the P2X2 and P2X3 receptor antagonists, suramin or pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS). These frequencies, number of bursts, and voltages were determined to maximise both AVP and OT release by electrical stimulations. Treatment of WT mouse NH with suramin/PPADS significantly reduced electrically‐stimulated AVP release. A similar inhibition by suramin was observed in electrically‐stimulated NH from P2X3 and P2X7 rKO mice but not P2X2/3 rKO mice, indicating that endogenous ATP facilitation of electrically‐stimulated AVP release is mediated primarily by the activation of the P2X2 receptor. Unexpectedly, electrically‐stimulated OT release from WT, P2X3, P2X2/3 and P2X7 rKO mice was potentiated by suramin, indicating nonpurinergic effects by this ‘selective’ antagonist. Nevertheless, these results show that sufficient endogenous ATP is released by bursts of action potentials to act at P2X2 receptors in a positive‐feedback mechanism to ‘differentially’ modulate neuropeptide release from central nervous system terminals.

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 (ICa) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, μ-opioid receptor (MOR)-mediated inhibition of ICa 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 ([Ca2+]i) in response to application of [d-Ala2-N-Me-Phe4,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 [Ca2+]i with BAPTA-AM completely blocked the inhibition of both ICa and high-K+-induced rises in [Ca2+]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 ICa and completely relieves MOR-mediated inhibition of high-K+-induced and DAMGO-induced rises in [Ca2+]i. Furthermore, antagonist concentrations of ryanodine completely blocked MOR-induced increases in [Ca2+]i and inhibition of ICa and high-K+-induced rises in [Ca2+]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 [Ca2+]i released by cADPr from ryanodine-sensitive stores.