Glucocorticoids rapidly inhibit oxytocin-stimulated adrenocorticotropin release from rat anterior pituitary cells, without modifying intracellular calcium transients

Glucocorticoid hormones suppress the secretion of ACTH evoked by secretagogues such as CRF and arginine vasopressin. In this study, we investigated the effects of glucocorticoids on ACTH release induced by oxytocin (OT) and on intracellular free calcium ion levels in corticotropes prepared from the adenohypophyses of female Wistar rats. Pulsatile additions of physiological concentration of OT (10 nM) to superfused anterior pituitary cells caused pulsatile ACTH release about 4-fold above basal secretion with similar peak amounts of ACTH during subsequent OT pulses. Exposure of the cells to corticosterone (100 nM) or to a selective glucocorticoid receptor agonist RU 28362 (100 nM) for 30 min suppressed OT-stimulated but not basal ACTH release by approximately 60%. Inhibition gradually disappeared during subsequent pulses of OT in the absence of corticosterone. Pretreatment with the selective antagonist RU 38486 (1 microM) completely blocked the inhibitory effect of corticosterone on OT-induced ACTH secretion. Changes in free cytosolic calcium levels in single cultured pituitary cells were measured using the calcium indicator Fura-2. OT caused calcium transients in corticotropes, which were identified by immunocytochemistry. They responded in a similar manner to a second OT stimulus when preincubated for 30 min with corticosterone (1 microM) or with RU 28362 (1 microM). Our data indicate that glucocorticoids, via glucocorticoid receptors, rapidly inhibit OT-stimulated ACTH secretion by corticotropes without affecting intracellular calcium transients due to OT. Therefore, we conclude that rapid inhibition of ACTH release by glucocorticoids interferes with cellular signal transduction beyond the step of calcium mobilization

Oxytocin at physiological concentrations evokes adrenocorticotropin (ACTH) release from corticotrophs by increasing intracellular free calcium mobilized mainly from intracellular stores. Oxytocin displays synergistic or additive effects on ACTH-releasing factor or arginine vasopressin-induced ACTH secretion, respectively

The potency of oxytocin (OT) in evoking ACTH secretion by isolated, superfused rat adenohypophyseal corticotrophs and its enhancement by CRF and arginine vasopressin (AVP) were analyzed. Each secretagogue effectively released ACTH from adenohypophyseal cells when added separately in pulsatile fashion in physiological concentrations based on hypophyseal portal blood (OT, 10 nM; AVP, 0.5 nM; CRF, 0.1 nM). OT released ACTH at concentrations as low as 1 nM. Moreover, a dose- response relationship up to 10 microM was revealed. Combinations of a constant amount of CRF (0.1 nM) with increasing concentrations of OT exerted a synergistic effect on ACTH release. In contrast, OT given in various concentrations in combination with AVP (0.5 nM) produced an additive effect on ACTH release. To study the mechanism of action of OT on ACTH secretion, cytosolic free calcium levels in single pituitary cells exposed to OT or AVP were measured using the calcium-sensitive fluorescent indicator Fura-2. Corticotrophs among mixed adenohypophyseal cell types in the primary cultures were identified by immunocytochemistry. More than 500 cells were individually stimulated with OT or AVP. Basal cytosolic free calcium levels ranged between 80- 130 nM free calcium. The addition of 100 nM OT or 1 microM AVP increased the cytosolic free calcium concentration within 3 sec to values ranging from 500-800 nM. An increase in intracellular calcium ranging from 200-500 nM due to OT could still be observed after extracellular calcium depletion. Taken together, our data demonstrate that physiological concentrations of OT stimulate ACTH secretion, independent of the other ACTH secretagogues, by mobilizing calcium mainly from intracellular stores

Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin

The intracellular action on exocytosis of botulinim A toxin and constituent chains was studied using permeabilized isolated nerve endings from the rat neural lobe. The release of the neuropeptide vasopressin was measured by radioimmunoassay. In the presence of the reducing agent dithiothreitol, the two-chain form of botulinum A toxin inhibited vasopressin release induced by 10 μM free calcium. Half maximal inhibition was obtained with 15 nM botulinum A toxin. In the absence of the heavy chain the light chain of the toxin strongly inhibited exocytosis with a half maximal effect of 2.5 nM. The inhibitory effects on secretion could be prevented by incubating the light chain with an immune serum against botulinum A toxin. The heavy chain of botulinum A toxin did not affect vasopressin release. However, it prevented the inhibitory effects of the light chain on stimulated exocytosis. It is concluded that botulinum A toxin inhibits the calcium-dependent step leading to exocytosis by interfering with a target present in the isolated and permeabilized nerve terminals. The functional domain of this neurotoxin, which is responsible for the inhibition of vasopressin release, is present in its light chain

Effects of the surgical excision of the sinus gland and eyestalk ablation on osmotic regulation

Sinus gland is a neurohaemal organ wherein hormones from different neurosecretory centres are stored. The sinus gland of Scylla serrata is well developed and macroscopically visible owing to its well known characteristic opacity and slightly bluishhue. It is located at the dorsal aspect of the junction between the medulla interna and the medulla terminalis in the eyestalk. Since it is a compact structure it is possible to remove the sinus gland from the eyestalk. There are two surgical procedures for the removal of the sinus gland. The first procedure involves the removal of the retinal portion of the eye cap and the other involves surgical excision without disturbing the eye cap and thereby the vision of the crab (Kleinholz, 1947)

Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin

The intracellular action on exocytosis of botulinim A toxin and constituent chains was studied using permeabilized isolated nerve endings from the rat neural lobe. The release of the neuropeptide vasopressin was measured by radioimmunoassay. In the presence of the reducing agent dithiothreitol, the two-chain form of botulinum A toxin inhibited vasopressin release induced by 10 μM free calcium. Half maximal inhibition was obtained with 15 nM botulinum A toxin. In the absence of the heavy chain the light chain of the toxin strongly inhibited exocytosis with a half maximal effect of 2.5 nM. The inhibitory effects on secretion could be prevented by incubating the light chain with an immune serum against botulinum A toxin. The heavy chain of botulinum A toxin did not affect vasopressin release. However, it prevented the inhibitory effects of the light chain on stimulated exocytosis. It is concluded that botulinum A toxin inhibits the calcium-dependent step leading to exocytosis by interfering with a target present in the isolated and permeabilized nerve terminals. The functional domain of this neurotoxin, which is responsible for the inhibition of vasopressin release, is present in its light chain

The light chain of tetanus toxin inhibits calcium-dependent vasopressin release from permeabilized nerve endings

The effects of tetanus toxin and its light and heavy chain subunits on vasopressin release were investigated in digitonin-permeabilized neurosecretory nerve terminals isolated from the neural lobe of the rat pituitary gland. Exocytosis was induced by challenging the permeabilized nerve endings with micromolar calcium concentrations. Tetanus toxin inhibited vasopressin release only in the presence of the reducing agent dithiothreitol. This effect was irreversible. The purified light chain of tetanus toxin strongly inhibited exocytosis in a dose-dependent manner with half-maximal effect at c. 10 nM. The action of the light chain was observed after only 2.5 min of preincubation. Separated heavy chain subunit had no effect on hormone secretion. Inhibition of vasopressin release could be prevented by preincubating the light chain of tetanus toxin with an immune serum against tetanus toxin. The data clearly demonstrate that in mammalian neurosecretory nerve endings tetanus toxin acts at a step downstream from the activation by Ca2+ of the exocytotic machinery and that the functional domain of this toxin is confined to its light chain

Exploring the functional domain and the target of the tetanus toxin light chain in neurohypophysial terminals

The tetanus toxin light chain blocks calcium induced vasopressin release from neurohypophysial nerve terminals. Here we show that histidine residue 233 within the putative zinc binding motif of the tetanus toxin light chain is essential for the inhibition of exocytosis, in the rat. The zinc chelating agent dipicolinic acid as well as captopril, an inhibitor of zinc-dependent peptidases, counteract the effect of the neurotoxin. Synthetic peptides, the sequences of which correspond to motifs present in the cytoplasmic domain of the synaptic vesicle membrane protein synaptobrevin 1 and 2, prevent the effect of the tetanus toxin light chain. Our results indicate that zinc bound to the zinc binding motif constitutes the active site of the tetanus toxin light chain. Moreover they suggest that cleavage of synaptobrevin by the neurotoxin causes the inhibition of exocytotic release of vasopressin from secretory granules

Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin

The intracellular action on exocytosis of botulinim A toxin and constituent chains was studied using permeabilized isolated nerve endings from the rat neural lobe. The release of the neuropeptide vasopressin was measured by radioimmunoassay. In the presence of the reducing agent dithiothreitol, the two-chain form of botulinum A toxin inhibited vasopressin release induced by 10 μM free calcium. Half maximal inhibition was obtained with 15 nM botulinum A toxin. In the absence of the heavy chain the light chain of the toxin strongly inhibited exocytosis with a half maximal effect of 2.5 nM. The inhibitory effects on secretion could be prevented by incubating the light chain with an immune serum against botulinum A toxin. The heavy chain of botulinum A toxin did not affect vasopressin release. However, it prevented the inhibitory effects of the light chain on stimulated exocytosis. It is concluded that botulinum A toxin inhibits the calcium-dependent step leading to exocytosis by interfering with a target present in the isolated and permeabilized nerve terminals. The functional domain of this neurotoxin, which is responsible for the inhibition of vasopressin release, is present in its light chain

Advances in the neurophysiology of magnocellular neuroendocrine cells

© 2020 British Society for Neuroendocrinology Hypothalamic magnocellular neuroendocrine cells have unique electrical properties and a remarkable capacity for morphological and synaptic plasticity. Their large somatic size, their relatively uniform and dense clustering in the supraoptic and paraventricular nuclei, and their large axon terminals in the neurohypophysis make them an attractive target for direct electrophysiological interrogation. Here, we provide a brief review of significant recent findings in the neuroplasticity and neurophysiological properties of these neurones that were presented at the symposium “Electrophysiology of Magnocellular Neurons” during the 13th World Congress on Neurohypophysial Hormones in Ein Gedi, Israel in April 2019. Magnocellular vasopressin (VP) neurones respond directly to hypertonic stimulation with membrane depolarisation, which is triggered by cell shrinkage-induced opening of an N-terminal-truncated variant of transient receptor potential vanilloid type-1 (TRPV1) channels. New findings indicate that this mechanotransduction depends on actin and microtubule cytoskeletal networks, and that direct coupling of the TRPV1 channels to microtubules is responsible for mechanical gating of the channels. Vasopressin neurones also respond to osmostimulation by activation of epithelial Na+ channels (ENaC). It was shown recently that changes in ENaC activity modulate magnocellular neurone basal firing by generating tonic changes in membrane potential. Both oxytocin and VP neurones also undergo robust excitatory synapse plasticity during chronic osmotic stimulation. Recent findings indicate that new glutamate synapses induced during chronic salt loading express highly labile Ca2+-permeable GluA1 receptors requiring continuous dendritic protein synthesis for synapse maintenance. Finally, recordings from the uniquely tractable neurohypophysial terminals recently revealed an unexpected property of activity-dependent neuropeptide release. A significant fraction of the voltage-dependent neurohypophysial neurosecretion was found to be independent of Ca2+ influx through voltage-gated Ca2+ channels. Together, these findings provide a snapshot of significant new advances in the electrophysiological signalling mechanisms and neuroplasticity of the hypothalamic-neurohypophysial system, a system that continues to make important contributions to the field of neurophysiology

Somato-dendritic vasopressin and oxytocin secretion in endocrine and autonomic regulation

Somato‐dendritic secretion was first demonstrated over 30 years ago. However, although its existence has become widely accepted, the function of somato‐dendritic secretion is still not completely understood. Hypothalamic magnocellular neurosecretory cells were among the first neuronal phenotypes in which somato‐dendritic secretion was demonstrated and are among the neurones for which the functions of somato‐dendritic secretion are best characterised. These neurones secrete the neuropeptides, vasopressin and oxytocin, in an orthograde manner from their axons in the posterior pituitary gland into the blood circulation to regulate body fluid balance and reproductive physiology. Retrograde somato‐dendritic secretion of vasopressin and oxytocin modulates the activity of the neurones from which they are secreted, as well as the activity of neighbouring populations of neurones, to provide intra‐ and inter‐population signals that coordinate the endocrine and autonomic responses for the control of peripheral physiology. Somato‐dendritic vasopressin and oxytocin have also been proposed to act as hormone‐like signals in the brain. There is some evidence that somato‐dendritic secretion from magnocellular neurosecretory cells modulates the activity of neurones beyond their local environment where there are no vasopressin‐ or oxytocin‐containing axons but, to date, there is no conclusive evidence for, or against, hormone‐like signalling throughout the brain, although it is difficult to imagine that the levels of vasopressin found throughout the brain could be underpinned by release from relatively sparse axon terminal fields. The generation of data to resolve this issue remains a priority for the field.http://wileyonlinelibrary.com/journal/jne2021-04-17hj2020Immunolog