Neurohypophysial Terminals Research Articles

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.

Chapter 12 Involvement of the brain oxytocin system in stress coping: interactions with the hypothalamo-pituitary-adrenal axis

In response to various ethologically relevant stressors, oxytocin is released not only from neurohypophysial terminals into the blood, but also within distinct brain regions, for example the hypothalamic supraoptic and paraventricular nuclei, the septum and the amygdala in dependence on the quality and intensity of the stressor. Thus, oxytocin secretory activity may accompany the response of the hypothalamo-pituitary-adrenal (HPA) axis to a given stressor. In the present chapter, I try to summarize our efforts to reveal the physiological significance of intracerebrally released oxytocin in rats with respect to the regulation of the HPA axis under basal and stress conditions as well as with respect to behavioural stress responses. The effects of oxytocin appear to depend on the brain region studied and the state of activity of the animal (basal versus stress). In order to reveal interactions between the oxytocin system and the HPA axis, preliminary results are presented pointing towards a differential action of glucocorticoids on intracerebral and peripheral oxytocin release.

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

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

Plasticity of neurohypophysial terminals with increased hormonal release during dehydration: ultrastructural and biochemical analyses.

Arginine vasopressin- (AVP) and oxytocin- (OXT) secreting magnocellular neurons undergo gross structural changes with chronic physiological stimulation. Here, we investigated subcellular aspects of plasticity in rat neurohypophysial terminals during dehydration. Ultrastructural analyses demonstrated that chronic dehydration by 2% NaCl drinking for 7 days significantly decreased the numbers of neurosecretory granules and microvesicles but not the numbers of mitochondria. Moreover, in dehydrated rats, terminals making neurovascular contacts enlarged, whereas terminals in apposition to astrocytes, i.e., neuroglial contacts, became smaller. Western blot analyses demonstrated significant decreases in the levels of F3 and Thy-1 together with those of AVP- and OXT-neurophysin, but the levels of synaptophysin, SNAP-25, and GAP-43 were unchanged. Both F3 and Thy-1 were recovered in the buffer-insoluble pellet, and phosphatidyl inositol-specific phospholipase C treatment released both molecules from the crude membrane fraction, indicating that they are attached to terminal membranes by glycosylphosphatidyl inositol anchors. Confocal microscopic observations demonstrated that F3 colocalized with Thy-1 in the same terminals of magnocellular neurons. In contrast, the level of calretinin, a Ca(2+) binding protein was significantly increased with chronic dehydration. Thus, the present results suggest that enhancement of neurovascular contacts results from rearrangement of terminal-astrocyte and terminal-vessel contacts rather than enlargement or sprouting of magnocellular terminals themselves. The down-regulation of F3 and Thy-1 may contribute to enhancement of neurovascular contacts that accompany increased peptide release during dehydration.

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.

Intracellular Ca 2+ channel immunoreactivity in neuroendocrine axon terminals

The concentration of neuroendocrine terminals in the neurohypophysis facilitates the identification and localization of Ca 2+ channel subtypes near neuroendocrine release sites. Immunoblots of rat neurohypophysial tissue identified the α 11.3, α 12.1, α 12.2, and α 12.3 Ca 2+ channel subunits. Immunofluorescence staining of axon terminal plasma membranes was weak, suggesting that Ca 2+ channels are dispersed. This contrasts with the highly punctate α 12.2 immunoreactivity in bovine chromaffin cells; the neurohypophysial terminals may therefore lack the specialized release zones found in those cells. Immunofluorescence and immunogold labeling identify dense core granule-like structures in the terminal cytoplasm containing multiple Ca 2+ channel types. Ca 2+ channels in internal membranes may play an important role in channel targeting and distribution in neuroendocrine cells.

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.

Tolerance to Acute Ethanol Inhibition of Peptide Hormone Release in the Isolated Neurohypophysis

Acute ethanol (EtOH) exposure reduces the evoked release of vasopressin (AVP) and oxytocin (OT) from excised neurohypophyses and from dissociated neurohypophysial terminals of the rat. Rats placed on a diet that maintained blood levels of 30 mM EtOH for 20 to 40 days developed tolerance to acute EtOH inhibition of release. In the presence of 10 mM EtOH, high (50 mM) K+-induced release of AVP from isolated neurohypophysial terminals of EtOH-naive rats was reduced by 77.7+/-1.4%, whereas in the chronic EtOH group, release was reduced by only 9.4+/-8.7%. Similar tolerance was evident during acute challenge with 75 mM EtOH, as well as for release of OT from isolated terminals. Animals treated with an intraperitoneal injection of EtOH and sacrificed 90 min postinjection did not exhibit the reduced EtOH inhibition of release from dissociated terminals during a 75 mM EtOH acute challenge. The altered component responsible for the tolerance to inhibition of release resides in the isolated terminal, because tolerance measured in vitro from intact neurohypophyses was similar to that seen in isolated terminals. The failure of EtOH-injected animals to exhibit reduced inhibition of release in response to an acute EtOH challenge indicates that short-term elevated blood alcohol level does not induce this tolerance. The finding of tolerance to EtOH-induced inhibition of release from the intact neurohypophysis and isolated terminals provides a physiological preparation in which to examine the molecular targets of acute drug action modified after chronic exposure to the drug.

Excitatory versus inhibitory modulation by ATP of neurohypophysial terminal activity in the rat.

Much is now known about the electrophysiological properties of the magnocellular neurones of the hypothalamus. Oxytocin neurones are characterized by an intermittent high frequency discharge during suckling that leads to the pulsatile release of oxytocin into the blood and to subsequent milk ejection. Vasopressin neurones are characterized by their asynchronous phasic activity (bursting) during maintained vasopressin release and the subsequent regulation of water balance. In both cases, it is the clustering of spikes, albeit with different time courses for each peptide, that facilitates hormone release. The mechanism underlying this differential facilitation is one of the major unanswered questions in neuroendocrinology. This paper considers recent evidence that indicates that ATP, co-secreted with vasopressin and oxytocin, may play a key role in the regulation of stimulus-secretion coupling in the neurohypophysis. The activity of the type (II) Ca2+-activated K+ (K(Ca)) channel found in the nerve terminals was significantly increased in the presence of ATP on the cytoplasmic side of the channel. Extracellular ATP, in contrast, inhibited the type II K(Ca) current in a dose-dependent manner. Thus, intracellular and extracellular ATP exert opposite effects on the type II K(Ca) channel of neurohypophysial terminals. Furthermore, ATP opens P2X2 channels to increase intracellular [Ca2+] in the nerve terminals and subsequent arginine vasopressin (AVP) release. In contrast, adenosine, acting via A1 receptors, specifically inhibits only the N-type Ca2+ channel, thus decreasing neuropeptide release. These multiple, conflicting effects of ATP and its metabolite adenosine could explain the patterns of AVP release observed during physiological stimulation in vivo.

An R-Type Ca2+Current in Neurohypophysial Terminals Preferentially Regulates Oxytocin Secretion

Multiple types of voltage-dependent Ca(2+) channels are involved in the regulation of neurotransmitter release (Tsien et al., 1991; Dunlap et al., 1995). In the nerve terminals of the neurohypophysis, the roles of L-, N-, and P/Q-type Ca(2+) channels in neuropeptide release have been identified previously (Wang et al., 1997a). Although the L- and N-type Ca(2+) currents play equivalent roles in both vasopressin and oxytocin release, the P/Q-type Ca(2+) current only regulates vasopressin release. An oxytocin-release and Ca(2+) current component is resistant to the L-, N-, and P/Q-type Ca(2+) channel blockers but is inhibited by Ni(2+). A new polypeptide toxin, SNX-482, which is a specific alpha(1E)-type Ca(2+) channel blocker (Newcomb et al., 1998), was used to characterize the biophysical properties of this resistant Ca(2+) current component and its role in neuropeptide release. This resistant component was dose dependently inhibited by SNX-482, with an IC(50) of 4.1 nM. Furthermore, SNX-482 did not affect the other Ca(2+) current types in these CNS terminals. Like the N- and P/Q-type Ca(2+) currents, this SNX-482-sensitive transient Ca(2+) current is high-threshold activated and shows moderate steady-state inactivation. At the same concentrations, SNX-482 blocked the component of oxytocin, but not of vasopressin, release that was resistant to the other channel blockers, indicating a preferential role for this type of Ca(2+) current in oxytocin release from neurohypophysial terminals. Our results suggest that an alpha(1E) or "R"-type Ca(2+) channel exists in oxytocinergic nerve terminals and, thus, functions in controlling only oxytocin release from the rat neurohypophysis.

Pulsed Laser Imaging of Ca2+Influx in a Neuroendocrine Terminal

The surge of Ca(2+) that triggers vesicle fusion is shaped by the distribution of Ca(2+) channels and the physical relationship between those channels and the exocytotic apparatus. Although channels and the release apparatus are thought to be tightly associated at fast synapses, the arrangement at neuroendocrine cells is less clear. The distribution of Ca(2+) influx near release sites is difficult to determine because of spatial and temporal limitations on Ca(2+) imaging techniques. We now present spatially resolved images of Ca(2+) influx into rat neuroendocrine terminals on a millisecond time scale. Images of voltage-dependent Ca(2+) influx into neurohypophysial terminals were captured after excitation of Ca(2+)-sensitive dyes with pulses of laser light lasting a fraction of a microsecond. Submembranous Ca(2+) increases were detected during the first millisecond of an evoked Ca(2+) tail current. Steep gradients of Ca(2+) were evident, with concentrations near the membrane reaching above 1 microM during a 30 msec depolarization. Ca(2+) influx appeared evenly distributed, even when diffusion was restricted with an exogenous Ca(2+) chelator. During longer depolarizations, mean and peak Ca(2+) concentrations reached an asymptote in parallel, suggesting that Ca(2+) binding proteins near the membrane rapidly buffer Ca(2+) and do not become saturated during prolonged influx. These data support the hypothesis that exocytosis is activated in these terminals by the summation of influx through multiple, randomly spaced Ca(2+) channels.

Differences in the Properties of Ionotropic Glutamate Synaptic Currents in Oxytocin and Vasopressin Neuroendocrine Neurons

Oxytocin (OT) and vasopressin (VP) hormone release from neurohypophysial terminals is controlled by the firing pattern of neurosecretory cells located in the hypothalamic supraoptic (SON) and paraventricular nuclei. Although glutamate is a key modulator of the electrical activity of both OT and VP neurons, a differential contribution of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) has been proposed to mediate glutamatergic influences on these neurons. In the present study we examined the distribution and functional properties of synaptic currents mediated by AMPARs and NMDARs in immunoidentified SON neurons. Our results suggest that the properties of AMPA-mediated currents in SON neurons are controlled in a cell type-specific manner. OT neurons displayed AMPA-mediated miniature EPSCs (mEPSCs) with larger amplitude and faster decay kinetics than VP neurons. Furthermore, a peak-scaled nonstationary noise analysis of mEPSCs revealed a larger estimated single-channel conductance of AMPARs expressed in OT neurons. High-frequency summation of AMPA-mediated excitatory postsynaptic potentials was smaller in OT neurons. In both cell types, AMPA-mediated synaptic currents showed inward rectification, which was more pronounced in OT neurons, and displayed Ca2+ permeability. On the other hand, NMDA-mediated mEPSCs of both cell types had similar amplitude and kinetic properties. The cell type-specific expression of functionally different AMPARs can contribute to the adoption of different firing patterns by these neuroendocrine neurons in response to physiological stimuli.

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.

Ethanol reduces the duration of single evoked spikes by a selective inhibition of voltage-gated calcium currents in acutely dissociated supraoptic neurons of the rat.

The effects of ethanol were studied on single evoked spikes recorded at 20 degrees C with the perforated-patch method in acutely dissociated rat supraoptic neurons. In seven out of eight neurons, ethanol (50 mM) significantly reduced the spike duration by selectively decreasing the decay time (82+/-2% of the control), leaving the amplitude and rise time unaffected. Resting potential and threshold did not change. Similarly, CdCl2 at a concentration of 100 microM, which blocks all voltage-activated calcium current in the supraoptic neurons, reduced the decay time of single evoked spikes (76+/-3% of the control, n=10) without modifying the other above-mentioned parameters. In addition, exposure to 100 microM CdCl2 prevented any subsequent effect of 50 mM ethanol (n = 5). Exposure to apamin (10 nM) and iberiotoxin (10 nM) did not have any effect on single evoked spikes. Because these concentrations are effective in blocking, respectively, small (SK) and large (BK) conductance calcium-dependent potassium channels in these neurons, this result shows that these currents are not involved in either the shaping of single evoked spikes or the actions of ethanol on spike shape. The sustained component of whole-cell recorded calcium current measured at -10 mV (hp -60 mV) was inhibited by ethanol in a dose-dependent manner, with a significant effect detectable at 25 mM. Exposure to 50 mM ethanol significantly reduced the sustained current to 70+/-5% of the control (n=12), without any apparent shift of the current-voltage relationship. Control exposure of the neurons to either 50 mM urea or 50 mM sucrose did not affect the voltage-gated calcium currents. We conclude that ethanol reduces the duration of single evoked spikes by a specific inhibition of voltage-activated calcium currents. The results suggest that, in addition to its direct effects on release of vasopressin and oxytocin from neurohypophysial terminals, ethanol could also affect hormonal release via changes in firing patterns arising in the cell bodies.

Analysis of angiotensin type 2 receptors in vasopressinergic neurons and pituitary in the rat

Previous functional studies indicated that an angiotensin type 2 (AT 2) receptor subtype may participate in the regulation of vasopressin release by angiotensin II (AngII). In the present study, AT 2 receptor-directed antiserum immunohistochemically detected AT 2 receptors within the hypothalamic paraventricular (PVN) and the supraoptic nuclei (SON) of the rat brain, more specifically, in identified vasopressinergic neurons. Considering the lack of AT 2 binding in the PVN and the SON using receptor autoradiography, we tested the hypothesis that these AT 2 receptors are transported to the posterior pituitary. Western blot analysis detected AT 2 immunoreactivity in the posterior pituitary. However, no AT 2 binding was detected in posterior pituitary membranes, and no AT 2 binding was detected with quantitative receptor autoradiography in the neurohypophysis. Thus, if AT 2 receptors are transported from the magnocellular vasopressin neurons to the posterior pituitary, their role in AngII regulation of vasopressin release at the neurohypophyseal terminals remains to be clarified.

Role of Q-type Ca2+ channels in vasopressin secretion from neurohypophysial terminals of the rat.

1. The nerve endings of rat neurohypophyses were acutely dissociated and a combination of pharmacological, biophysical and biochemical techniques was used to determine which classes of Ca2+ channels on these central nervous system (CNS) terminals contribute functionally to arginine vasopressin (AVP) and oxytocin (OT) secretion. 2. Purified neurohypophysial plasma membranes not only had a single high-affinity binding site for the N-channel-specific omega-conopeptide MVIIA, but also a distinct high-affinity site for another omega-conopeptide (MVIIC), which affects both N- and P/Q-channels. 3. Neurohypophysial terminals exhibited, besides L- and N-type currents, another component of the Ca2+ current that was only blocked by low concentrations of MVIIC or by high concentrations of omega-AgaIVA, a P/Q-channel-selective spider toxin. 4. This Ca2+ current component had pharmacological and biophysical properties similar to those described for the fast-inactivating form of the P/Q-channel class, suggesting that in the neurohypophysial terminals this current is mediated by a 'Q'-type channel. 5. Pharmacological additivity studies showed that this Q-component contributed to rises in intraterminal Ca2+ concentration ([Ca2+]i) in only half of the terminals tested. 6. Furthermore, the non-L- and non-N-component of Ca(2+)-dependent AVP release, but not OT release, was effectively abolished by the same blockers of Q-type current. 7. Thus Q-channels are present on a subset of the neurohypophysial terminals where, in combination with N- and L-channels, they control AVP but not OT peptide neurosecretion.

Immunocytochemical evidence for the presence of vasopressin in intermediate sized neurosecretory granules of solitary neurohypophyseal terminals in the homozygous brattleboro rat

A single base deletion (delta G) in the vasopressin gene is the cause of diabetes insipidus in the homozygous Brattleboro rat (di/di). The resulting frameshift leads to the expression of an aberrant vasopressin precursor which is unable to enter the secretory pathway, thereby preventing vasopressin biosynthesis. In a small number of solitary magnocellular hypothalamic neurons within the supraoptic and paraventricular nuclei, the reading frame is restored by a dinucleotide (delta GA) frameshift mutation, at two separate GAGAG motifs downstream of the original G-deletion. This results in two + 1 di-vasopressin precursors that are still partially mutated within the neurophysin region. The present study provides immunocytochemical evidence which demonstrates that, within magnocellular solitary neurons of the supraoptic and paraventricular nuclei of the di/di rat, the + 1 di-vasopressin precursors can enter the secretory pathway followed by their enzymatic processing into vasopressin during axonal transport to the neural lobe. However, the cellular characteristics of biosynthesis are different from those of wild-type rats. Immunoelectron microscopical localization of vasopressin gene products in the neural lobe of did/di rats revealed their presence in neurosecretory granules, the diameter of which is intermediate (116 nm) between those of the neurosecretory granules in the di/di (80-100 nm) and wild-type (160 nm) rats.

Micromolar 4-aminopyridine enhances invasion of a vertebrate neurosecretory terminal arborization: optical recording of action potential propagation using an ultrafast photodiode-MOSFET camera and a photodiode array.

Modulation of the amount of neuropeptide released from a neurosecretory tissue may be achieved by different means. These include alterations in the quantity secreted from each active nerve terminal or in the actual number of terminals activated. From the vertebrate hypothalamus, magnocellular neurons project their axons as bundles of fibers through the median eminence and infundibular stalk to arborize extensively and terminate in the neurohypophysis, where the neurohypophysial peptides and proteins are released into the circulation by a Ca-dependent mechanism. Elevating [Ca2+]o increases the magnitude of an intrinsic optical change in the neurohypophysial terminals that is intimately related to the quantity of neuropeptide released. Similarly, the addition of micromolar concentrations of 4-aminopyridine to the bathing solution enhances this change in large angle light scattering. However, we show here that, while these effects are superficially similar, they reflect different mechanisms of action. Evidence from intrinsic optical signals (light scattering) and extrinsic (potentiometric dye) absorption changes suggests that calcium increases the amount of neuropeptide released from each active terminal in the classical manner, while 4-aminopyridine exerts its secretagogue action by enhancing the invasion of action potentials into the magno-cellular neuron's terminal arborization, increasing the actual number of terminals activated. Physiologically, electrical invasion of the complex terminal arborization in the neurohypophysis may represent an extremely sensitive control point for modulation of peptide secretion. This would be especially effective in a neurohaemal organ like the posterior pituitary, where, in contrast with a collection of presynaptic terminals, the precise location of release is less important than the quantity released.

Ba2+ ions evoke two kinetically distinct patterns of exocytosis in chromaffin cells, but not in neurohypophysial nerve terminals

The coupling between divalent cations and exocytosis of large dense-cored vesicles (LDCV) was studied with capacitance-detection techniques in nerve terminals of the rat neurohypophysis (NHP) and bovine chromaffin cells. Ba2+ substitution for Ca2+ produced kinetically distinct responses in the two preparations. In NHP terminals, Ba2+ ions behave as weak substitutes for Ca2+. Exocytotic events occur principally during depolarizing pulses, i.e., events are "stimulus-coupled" to Ba2+ entry through voltage-gated Ca2+ channels. Stimulus-coupled exocytosis apparently requires elevated submembrane cation concentrations that dissipate rapidly on hyperpolarization-induced Ca(2+)-channel closure. Intracellular dialysis of NHP terminals with Ba2+ does not evoke exocytosis, nor does it interfere with depolarization-evoked Ca2+ influx and exocytosis. In chromaffin cells, Ba2+ ions evoke a small quantity of stimulus-coupled secretion, but the dominant response is an additional pronounced poststimulus capacitance increase that outlasts channel closures by 20-50 sec. "Stimulus-decoupled" exocytosis is slow (approximately 25-40 fF/sec) compared with Ca(2+)-evoked stimulus-coupled exocytosis (approximately 1000 fF/sec). Decoupled secretion is not attributable to Ba2+ displacement of intracellular Ca2+ ions, because it is insensitive to 10 mM EGTA or thapsigargin. Slow exocytosis is initiated by inclusion of Ba2+ ions in the recording pipette and continues steadily for 5-12 min, producing a total increase of several thousand fF, which ultimately doubles or triples the original cell-surface area. We propose that two pathways of regulated exocytosis with distinct kinetics and divalent cation sensitivity exist in chromaffin cells. Only a single kinetic pattern is detected in NHP terminals, suggesting that mechanisms for secretion are not universally distributed in excitable cells.