Sonia Ortiz-Miranda

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.

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.

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.

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 µ‐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 AVPs 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 Ca2+ 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 β‐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. J. Cell. Physiol. 225: 276–288, 2010.

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 (EC50 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 (IC50 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 EC50, 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.

Functional ryanodine receptors in the membranes of neurohypophysial secretory granules

Localized calcium release from ryanodine-sensitive stores in large dense core vesicles may modulate secretion of neuropeptides from neurohypophysial terminals.

Purinergic receptor types in the hypothalamic-neurohypophysial system

Many different types of purinergic receptors are present in the hypothalamic‐neurohypophysial system (HNS), which synthesises 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 knockouts, 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, whereas 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 hypothesise 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.