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Day/night cycle controls neurogenesis; melatonin released from pineal gland in darkness stimulates intracellular Ca 2+ dynamics thus decreasing proliferation of neural stem cells. In the daylight intracellular Ca 2+ activity subsides, which stimulates neural stem cells division and increases generation of newborn neurones., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca 2+ concentration ([Ca 2+ ] i ) remain unknown. We analyzed mechanisms regulating resting [Ca 2+ ] i in dissociated rat melanotrophs by Ca 2+ -imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca 2+ channels (VGCCs) as well as removal of extracellular Ca 2+ resulted in a rapid and reversible decrease in [Ca 2+ ] i , indicating constitutive Ca 2+ influx through L-type VGCCs. Reduction of extracellular Na + concentration (replacement with NMDG + ) similarly decreased resting [Ca 2+ ] i . When cells were champed at -80 mV, decrease in the extracellular Na + resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na + caused hyperpolarisation. The holding current shifted in negative direction when extracellular K + concentration was increased from 5 mM to 50 mM in the presence of K + channel blockers, Ba 2+ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca 2+ ] i . These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca 2+ ] i in rat melanotrophs., (Copyright © 2019. Published by Elsevier Ltd.)

The magnocellular vasopressin (AVP) and oxytocin (OT) neurones exhibit specific electrophysiological behaviour, synthesise AVP and OT peptides and secrete them into the neurohypophysial system in response to various physiological stimulations. The activity of these neurones is regulated by the very same peptides released either somato-dendritically or when applied to supraoptic nucleus (SON) preparations in vitro. The AVP and OT, secreted somato-dendritically (i.e. in the SON proper) act through specific autoreceptors, induce distinct Ca(2+) signals and regulate cellular events. Here, we demonstrate that about 70% of freshly isolated individual SON neurones from the adult non-transgenic or transgenic rats bearing AVP (AVP-eGFP) or OT (OT-mRFP1) markers, produce distinct spontaneous [Ca(2+)]i oscillations. In the neurones identified (through specific fluorescence), about 80% of AVP neurones and about 60% of OT neurones exhibited these oscillations. Exposure to AVP triggered [Ca(2+)]i oscillations in silent AVP neurones, or modified the oscillatory pattern in spontaneously active cells. Hyper- and hypo-osmotic stimuli (325 or 275 mOsmol/l) respectively intensified or inhibited spontaneous [Ca(2+)]i dynamics. In rats dehydrated for 3 or 5days almost 90% of neurones displayed spontaneous [Ca(2+)]i oscillations. More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats were oscillatory vs. about 44% (OT-mRFP1 neurones) in virgins. Together, these results unveil for the first time that both AVP and OT neurones maintain, via Ca(2+) signals, their remarkable intrinsic in vivo physiological properties in an isolated condition., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Isolated supraoptic neurones generate spontaneous [Ca(2+)]i oscillations in isolated conditions. Here we report in depth analysis of the contribution of plasmalemmal ion channels (Ca(2+), Na(+)), Na(+)/Ca(2+) exchanger (NCX), intracellular Ca(2+) release channels (InsP3Rs and RyRs), Ca(2+) storage organelles, plasma membrane Ca(2+) pump and intracellular signal transduction cascades into spontaneous Ca(2+) activity. While removal of extracellular Ca(2+) or incubation with non-specific voltage-gated Ca(2+) channel (VGCC) blocker Cd(2+) suppressed the oscillations, neither Ni(2+) nor TTA-P2, the T-type VGCC blockers, had an effect. Inhibitors of VGCC nicardipine, ω-conotoxin GVIA, ω-conotoxin MVIIC, ω-agatoxin IVA (for L-, N-, P and P/Q-type channels, respectively) did not affect [Ca(2+)]i oscillations. In contrast, a specific R-type VGCC blocker SNX-482 attenuated [Ca(2+)]i oscillations. Incubation with TTX had no effect, whereas removal of the extracellular Na(+) or application of an inhibitor of the reverse operation mode of Na(+)/Ca(2+) exchanger KB-R7943 blocked the oscillations. The mitochondrial uncoupler CCCP irreversibly blocked spontaneous [Ca(2+)]i activity. Exposure of neurones to Ca(2+) mobilisers (thapsigargin, cyclopiazonic acid, caffeine and ryanodine); 4-aminopyridine (A-type K(+) current blocker); phospholipase C and adenylyl cyclase pathways blockers U-73122, Rp-cAMP, SQ-22536 and H-89 had no effect. Oscillations were blocked by GABA, but not by glutamate, apamin or dynorphin. In conclusion, spontaneous oscillations in magnocellular neurones are mediated by a concerted action of R-type Ca(2+) channels and the NCX fluctuating between forward and reverse modes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Adherent, fibroblastic cells from different tissues are thought to contain subsets of tissue-specific stem/progenitor cells (often called mesenchymal stem cells). These cells display similar cell surface characteristics based on their fibroblastic nature, but also exhibit differences in molecular phenotype, growth rate, and their ability to differentiate into various cell phenotypes. The mechanisms underlying these differences remain poorly understood. We analyzed Ca(2+) signals and membrane properties in rat adipose-derived stromal cells (ADSCs) and bone marrow stromal cells (BMSCs) in basal conditions, and then following a switch into medium that contains factors known to modify their character. Modified ADSCs (mADSCs) expressed L-type Ca(2+) channels whereas both L- and P/Q- channels were operational in mBMSCs. Both mADSCs and mBMSCs possessed functional endoplasmic reticulum Ca(2+) stores, expressed ryanodine receptor-1 and -3, and exhibited spontaneous [Ca(2+)]i oscillations. The mBMSCs expressed P2X7 purinoceptors; the mADSCs expressed both P2X (but not P2X7) and P2Y (but not P2Y1) receptors. Both types of stromal cells exhibited [Ca(2+)]i responses to vasopressin (AVP) and expressed V1 type receptors. Functional oxytocin (OT) receptors were, in contrast, expressed only in modified ADSCs and BMSCs. AVP and OT-induced [Ca(2+)]i responses were dose-dependent and were blocked by their respective specific receptor antagonists. Electrophysiological data revealed that passive ion currents dominated the membrane conductance in ADSCs and BMSCs. Medium modification led to a significant shift in the reversal potential of passive currents from -40 to -50mV in cells in basal to -80mV in modified cells. Hence membrane conductance was mediated by non-selective channels in cells in basal conditions, whereas in modified medium conditions, it was associated with K(+)-selective channels. Our results indicate that modification of ADSCs and BMSCs by alteration in medium formulation is associated with significant changes in their Ca(2+) signaling and membrane properties., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Stem cells (SCs) of different origins have brought hope as potential tools for the treatment of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Amyotrophic Lateral Sclerosis. Calcium signalling plays a key role in SC differentiation and proliferation, and dysregulation of Ca(2+) homeostasis may instigate pathological scenarios. Currently, the role of ion channels and receptors in SCs is not fully understood. In the recent years, we found that (i) the pre-differentiation of human embryonic SCs (hESCs) led to the activation of Ca(2+) signalling cascades and enhanced the functional activities of these cells, (ii) the Ca(2+) homeostasis and the physiological properties of hESC-derived neural precursors (NPs) changed during long term propagation in vitro, (iii) differentiation of NPs derived from human induced pluripotent SCs affects the expression of ion channels and receptors, (iv) these neuronal precursors exhibited spontaneous activity, indicating that their electrophysiological and Ca(2+) handling properties are similar to those of mature neurones, and (v) in mesenchymal SCs isolated from the adipose tissue and bone marrow of rats the expression profile of ion channels and receptors depends not only on the differentiation conditions but also on the source from which the cells were isolated, indicating that the fate and functional properties of the differentiated cells are driven by intrinsic mechanisms. Together, identification and assignment of a unique ion channel and a Ca(2+) handling footprint for each cell type would be necessary to qualify them as physiologically suitable for medical research, drug screening, and cell therapy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Neurones in the supraoptic nucleus (SON) of the hypothalamus possess intrinsic osmosensing mechanisms, which are lost in transient receptor potential vanilloid 1 (Trpv1)-knock-out mice. The molecular nature of the osmosensory mechanism in SON neurones is believed to be associated with the N-terminal splice variant of Trpv1, although their entire molecular structures have not been hitherto identified. In this study, we sought for TRPV1-related molecules and their function in the rat SON. We performed RT-PCR and immunohistochemistry to detect TRPV1-related molecules in the SON, and patch-clamp and imaging of the cytosolic Ca(2+) concentration ([Ca(2+)]i) to measure responses to osmolality changes and TRPV-related drugs in acutely dissociated SON neurones of rats. RT-PCR analysis revealed full-length Trpv1 and a new N-terminal splice variant, Trpv1_SON (LC008303) in the SON. Positive immunostaining was observed using an antibody against the N-terminal portion of TRPV1 in arginine vasopressin (AVP)-immunoreactive neurones, but not in oxytocin (OT)-immunoreactive neurones. Approximately 20% of SON neurones responded to mannitol (50 mM) with increased action potential firing, inward currents, and [Ca(2+)]i mobilization. Mannitol-induced responses were observed in AVP neurones isolated from AVP-eGFP transgenic rats and identified by GFP fluorescence, but not in OT neurones isolated from OT-mRFP transgenic rats and identified by RFP fluorescence. The mannitol-induced [Ca(2+)]i responses were reversibly blocked by the non-selective TRPV antagonist, ruthenium red (10 μM) and the TRPV1 antagonists, capsazepine (10 μM) and BCTC (10 μM). Although the TRPV1 agonist, capsaicin (100 nM) evoked no response at room temperature, it triggered cationic currents and [Ca(2+)]i elevation when the temperature was increased to 36°C. These results suggest that AVP neurones in the rat SON possess functional full-length TRPV1. Moreover, differences between the responses to capsaicin or hyperosmolality obtained in rat SON neurones and those obtained from dorsal root ganglion neurones or TRPV1-expressing cells indicate that the osmoreceptor expressed in the SON may be a heteromultimer in which TRPV1 is co-assembled with some other, yet unidentified, molecules., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

This special issue on Ca(2+) signalling in neuroendocrine cells is an opportunity to assess, through a range of first-class review articles, the complex world of endocrine signalling, a complexity that is probably best captured by calling it "diversity in unity". The unity comes from the fact that all the endocrine cells are excitable cells, able to generate action potentials and are using Ca(2+) as an essential informational molecule, coupling cell stimulation with the activation of secretion, through the exocytotic process. The 'diversity' element, illustrated by almost all the reviews, stems from the modalities employed to achieve the increase in cytosolic Ca(2+) signal, the balance between the participation of Ca(2+) entry through the plasma membrane voltage-operated Ca(2+) channels and the release of Ca(2+) from intracellular Ca(2+) stores, and the cross-talk between the Ca(2+) and cyclic AMP signalling pathways., (Copyright © 2012. Published by Elsevier India Pvt Ltd.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Every cell or neuronal type utilizes its own specific organization of its Ca(2+) homeostasis depending on its specific function and its physiological needs. The magnocellular neurones, with their somata situated in the supraoptic and paraventricular nuclei of the hypothalamus and their nerve terminals populating the posterior hypophysis (neural lobe) are a typical and classical example of a neuroendocrine system, and an important experimental model for attempting to understand the characteristics of the neuronal organization of Ca(2+) homeostasis. The magnocellular neurones synthesize, in a cell specific manner, two neurohormones: arginine-vasopressin (AVP) and oxytocin (OT), which can be released, in a strict Ca(2+)-dependent manner, both at the axonal terminals, in the neural lobe, and at the somatodendritic level. The two types of neurones show also distinct type of bioelectrical activity, associated with specific secretory patterns. In these neurones, the Ca(2+) homeostatic pathways such as the Na(+)/Ca(2+) exchanger (NCX), the endoplasmic reticulum (ER) Ca(2+) pump, the plasmalemmal Ca(2+) pump (PMCA) and the mitochondria are acting in a complementary fashion in clearing Ca(2+) loads that follow neuronal stimulation. The somatodendritic AVP and OT release closely correlates with intracellular Ca(2+) dynamics. More importantly, the ER Ca(2+) stores play a major role in Ca(2+) homeostatic mechanism in identified OT neurones. The balance between the Ca(2+) homeostatic systems that are in the supraoptic neurones differ from those active in the terminals, in which mainly Ca(2+) extrusion through the Ca(2+) pump in the plasma membrane and uptake by mitochondria are active. In both AVP and OT nerve terminals, no functional ER Ca(2+) stores can be evidenced experimentally. We conclude that the physiological significance of the complexity of Ca(2+) homeostatic mechanisms in the somatodendritic region of supraoptic neurones and their terminals can be multifaceted, attributable, in major part, to their specialized electrical activity and Ca(2+)-dependent neurohormone release., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Multiple mechanisms that maintain Ca(2+) homeostasis and provide for Ca(2+) signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca(2+) clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca(2+) homeostatic molecules on cytosolic Ca(2+) ([Ca(2+)](i)) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca(2+)](i) dynamics. When bathing the cells in a Na(+)-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca(2+)-ATPase (PMCA), La(3+), all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca(2+)](i) transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca(2+) transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca(2+) homeostatic pathways, the Na(+)/Ca(2+) exchanger, the endoplasmic reticulum Ca(2+) pump, the plasmalemmal Ca(2+) pump and mitochondria, are complementary in actively clearing Ca(2+) from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca(2+)](i) dynamics; (iii) there is (are) Ca(2+) clearance mechanism(s) distinct from the four outlined above; and (iv) Ca(2+) homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

NGF is a candidate for a pathogenic mediator of neuropathic pain after nerve injury and inflammation. It has been reported that adult rat dorsal root ganglion (DRG) neurons cultured in the presence of NGF at 100 ng/ml generate spontaneous action potentials. However, it is unclear what types of subpopulation of DRG neurons are affected by NGF and how the intracellular Ca(2+) concentration ([Ca(2+)](i)) changes in such neurons. To elucidate these points, we measured [Ca(2+)](i) in adult rat DRG neurons cultured with or without NGF. [Ca(2+)](i) fluctuated spontaneously in the absence of any stimuli in subpopulations of NGF-treated neurons, but such fluctuations were not observed in all NGF-untreated neurons. NGF-induced [Ca(2+)](i) fluctuations were inhibited by decreases in extracellular Na(+) concentration, TTX and Lidocaine, suggesting that spontaneous action potentials provoked the [Ca(2+)](i) fluctuation. NGF-induced [Ca(2+)](i) fluctuation was observed in small and medium sized neurons and in Capsaicin-sensitive neurons more frequently than in Capsaicin-non-responsive neurons. These results suggest that NGF acted on the nociceptive neurons and made them hyperexcitable to generate spontaneous action potentials and spontaneous [Ca(2+)](i) fluctuations. The [Ca(2+)](i) fluctuation induced by NGF may play some role in the regulation of membrane excitability of nociceptive sensory neurons and neuropathic pain.

Neuroactive steroids synthesized from the brain or peripheral sources are called neurosteroids. Beside their common nuclear effects, they are considered to be potent neuromodulators, acting rapidly mainly in a non-genomic manner, either through allosteric regulation of ionic channels, or through membrane-bound steroid receptors. In contrast to the situation in the adult, the neurotransmitter GABA is excitatory during development and plays a trophic role, in particular inducing calcium signals necessary for the regulation of excitability and neuronal maturation. We demonstrated that the primary metabolite of progesterone (Proges), allopregnanolone (Allo), evoked a robust Ca(2+) influx in foetal hypothalamic neurons and in postnatal supraoptic nucleus (SON) neurons. In the latter, this led to oxytocin and arginine vasopressin release. Interestingly, these responses were GABA(A) and oxytocin-receptor-dependent. Allo is a well-known positive allosteric modulator of GABA(A) receptors. It is noteworthy that two other steroids, Proges and 17-beta-estradiol, displayed the same effect on Ca(2+) and oxytocin release but to a lesser extent. Importantly, no effect was observed in adult neurons from the SON, or in neurohypophysial axon terminals, regardless of the stage. The molecular mechanisms of the neurosteroid actions are multifaceted and depend on the type of cells, and are thus extremely interesting and challenging. In the peripheral nervous system, Allo and Proges surprisingly inhibited the GABA-induced Ca(2+) increases in embryonic dorsal root ganglion neurons. We propose that this rapid, reversible and dose-dependent phenomenon (at very low concentrations) was mediated by membrane Proges receptors, since transcripts for a newly discovered receptor protein, 25-Dx, were detected in our model. Recently, novel families of membrane steroid receptors, activating intracellular-signalling pathways such as MAP kinases, have been identified and described. This opens new perspectives to understand the intracellular machinery involved in the interaction between neuropeptides and neurosteroids, two major regulators of hypothalamo-neurohypophysial system development.

The non-genomic role of neuroactive steroids on [Ca2+]i transients induced by GABA receptor activation was investigated in cultured dorsal root ganglia (DRG) neurones at embryonic stage E13. [Ca2+]i measurements were performed with Fura-2 fast fluorescence microfluorimetry. Application of the GABAA receptor agonist muscimol (Musci) evoked an increase in [Ca2+]i, confirming the excitatory effect of GABA at this embryonic stage. The muscimol-induced [Ca2+]i response was inhibited by progesterone (Proges) and its primary metabolite allopregnanolone (Allo) in a rapid, reversible and dose-dependent manner. These calcium transients were suppressed in the absence of external Ca2+ or in the presence of Ni2+ + Cd2+ suggesting an involvement of voltage-activated Ca2+ channels. In contrast, none of these steroids affected the resting [Ca2+]i nor exhibited any inhibitory effect on 50 mM KCl-induced [Ca2+]i increases. In view of the well-established potentiation of GABAA receptor by direct binding of neurosteroids, the inhibitory effects described in this study seem to involve distinct mechanisms. This new inhibitory effect of progesterone is observed at low and physiological concentrations, is rapid and independent of RU38486, an antagonist of the classic progesterone receptor, probably involving a membrane receptor. Using RT-PCR, we demonstrated the expression of progesterone receptor membrane component 1 (Pgrmc1), encoding 25-Dx, a membrane-associated progesterone binding protein in DRG neurones at different stages of development. In conclusion, we describe for the first time a rapid effect of progestins on embryonic DRG neurones involving an antagonistic effect of progesterone and allopregnanolone on GABAA receptors.

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca(2+) stores, via both ryanodine-sensitive and IP(3)-sensitive intracellular Ca(2+) channels control [Ca(2+)](i) homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca(2+)](i) increased progressively from days 1-7 in culture. These responses were blocked by ryanodine and nicardipine but not by omega-conotoxin-GVIA or omega-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Ca(v)1 Ca(2+) channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca(2+) oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca(2+) stores from the endoplasmic reticulum, induced an increase in [Ca(2+)](i) in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5-6 days in vitro. Xestospongin-C, a membrane-permeable IP(3) receptor inhibitor blocked the CPA-induced [Ca(2+)](i) response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP(3) Ca(2+) channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca(2+)](i) homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca(2+) channel responsible for a Ca(2+)-induced Ca(2+) release in embryonic motoneurons following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels.

The importance of intracellular calcium ([Ca2+]i) in the release of vasopressin (AVP) and oxytocin from the central nervous system neurohypopyhysial nerve terminals has been well-documented. To date, there is no clear understanding of Ca2+ clearance mechanisms and their interplay with transmembrane Ca2+ entry, intracellular [Ca2+]i transients, cytoplasmic Ca2+ stores and hence the release of AVP at the level of a single nerve terminal. Here, we studied the mechanism of Ca2+ clearance in freshly isolated nerve terminals of the rat neurohypophysis using Fura-2 Ca2+ imaging and measured the release of AVP by radioimmuno assay. An increase in the K+ concentration in the perfusion solution from 5 to 50 mM caused a rapid increase in [Ca2+]i and AVP release. Returning K+ concentration to 5 mM led to rapid restoration of both responses to basal level. The K+-evoked [Ca2+]i and AVP increase was concentration-dependent, reliable, and remained of constant amplitude and time course upon successive applications. Extracellular Ca2+ removal completely abolished the K+-evoked responses. The recovery phase was not affected upon replacement of NaCl with sucrose or drugs known to act on intracellular Ca2+ stores such as thapsigargin, cyclopiazonic acid, caffeine or a combination of caffeine and ryanodine did not affect either resting or K+-evoked [Ca2+]i or AVP release. By contrast, the plasma membrane Ca2+ pump inhibitor, La3+, markedly slowed down the recovery phase. The mitochondrial respiration uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), slightly but significantly increased the basal [Ca2+]i, and also slowed down the recovery phase of both [Ca2+]i and release responses. In conclusion, we show in nerve terminals that (i) Ca2+ extrusion through the Ca2+ pump in the plasma membrane plays a major role in the Ca2+ clearance mechanisms of (ii) Ca2+ uptake by mitochondria also contributes to the Ca2+ clearance and (iii) neither Na+/Ca2+ exchangers nor Ca2+ stores are involved in the Ca2+ clearance or in the maintenance of basal [Ca2+]i or release of AVP.

The interactions of the dendritically released neuropeptides vasopressin and oxytocin with co-released neuroactive substances such as opioids and nitric oxide are reviewed. Endogenous opioids regulate magnocellular neurons at the level of the supraoptic nucleus and the relationship of dendritically released peptides and co-released opioids seems to be dependent on the stimulus given and the physiological state of the animal. Nitric oxide has a prominent inhibitory action on supraoptic neurons and these actions are predominantly mediated indirectly by GABA inputs. The role of these co-released neuroactive substances in differentially regulated release of neuropeptides from dendrites versus distant axon terminals has to be determined in more detail. A picture emerges in which release of vasopressin and oxytocin from different anatomical compartments of a single neuron may arise from different intracellular secretory pools and their preparation before release.

The consequences of purinoceptor activation on calcium signalling, inositol phosphate metabolism, protein secretion and the actin cytoskeleton were demonstrated in the WRK-1 cell line. Extracellular ATP was used as a secretagogue to induce a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)), acting via P2x purinergic receptors, which causes actin skeleton disaggregation and protein secretion. ATP bound specifically to purinergic receptors, with Ki of 0.8 microM. The magnitude order for binding of different nucleotides was alpha beta-Met-ATP >or= dATPalphaS > ATP >or= ADP > UTP > AMP > suramin. No increase in inositol phosphates (IPs) was observed after ATP application suggesting that the purinergic sites in WRK-1 cells are not of a P2y type. ATP (1-100 microM) caused a concentration-dependent increase in [Ca(2+)](i)(EC(50)= 30 microM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca(2+)](e)) was reduced to 100 nM. A non-specific purinergic antagonist, suramin, reversibly inhibited the ATP-response suggesting that ATP is able to bind to P2x purinergic sites to trigger Ca(2+) entry and increase of [Ca(2+)](i). ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca(2+)](e). Similarly, alpha,beta-Met-ATP, a potent P2x agonist also stimulated Ca(2+) mobilization, actin network destructuration, and protein release. In the isolated rat neurohypophysial nerve terminals, ATP was shown to act as a physiological stimulus for vasopressin release via Ca(2+) entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is also able to induce actin disaggregation by a Ca(2+) dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis., (Copyright 2001 Harcourt Publishers Ltd.)

Intracellular Ca2+ ([Ca2+]i) was measured in single human epithelial intestinal HT-29-D4 cells with the Ca2+ probe Fura-2 and digital imaging microscopy. Treatment of these cells with HIV-1 surface envelope glycoprotein gp120 (or a soluble form of its precursor gp160) induced an important increase of [Ca2+]i. This effect was abolished by preincubation of the viral glycoprotein with neutralizing antibodies specific for the V3 domain of gp120. These antibodies inhibited the binding of both gp120 and gp160 to galactosylceramide (GalCer), the alternative HIV-1 receptor in HT-29-D4 cells. Moreover, treatment of HT-29-D4 cells with an anti-GalCer mAb induced an increase in [Ca2+]i and rendered the cells insensitive to HIV-1 glycoprotein stimulation. The calcium response resulted from release of Ca2+ from caffeine-sensitive intracellular stores. Finally, the viral glycoprotein specifically abrogated the calcium response to the neuropeptide agonist neurotensin, a stimulator of chloride secretion via inositol trisphosphate-mediated calcium mobilization. Reciprocally, after neurotensin stimulation, the cells did not respond to gp120, showing that neurotensin and gp120 stimulate a common pathway of [Ca2+]i mobilization. These results suggest that HIV-1 may directly alter ion secretion in the intestine and thus be the causative agent of the watery diarrhea associated with HIV-1 infection.

The secretion of corticotropin by perfused rat anterior pituitary cell columns was studied. Forty-one residue corticotropin releasing factor, vasopressin and high extracellular KC1 all stimulated the secretion of corticotropin. The hormonal response to corticotropin-releasing factor (10(-10) mol/l), vasopressin (10(-9) mol/l) as well as KC1 (48 mmol/l) was reduced by membrane permeant analogs of cGMP, such as 8-BrcGMP and dibutyryl-cGMP. The 8-BrcGMP analog (10(-5) mol/l) inhibited corticotropin release in response to corticotropin-releasing factor by 30%, that to vasopressin by 70%, and that to KCl by 50%. Atriopeptin1-28 (10(-8) and 10(-7) mol/l), a peptide known to activate membrane-bound guanylate cyclase in the anterior pituitary gland, decreased the release of corticotropin induced by vasopressin to about 30% of control. Similarly, activators of soluble guanylate cyclase, such as glyceryltrinitrate and sodium nitroprusside (10(-5) mol/l) inhibited vasopressin-stimulated corticotropin release by 60%. In conclusion, the data show that purported activators of particulate and soluble guanylate cyclase, as well as derivatives of cGMP itself are strong inhibitors of secretagogue-induced corticotropin release by corticotroph cells of the anterior pituitary gland.

The binding of omega-conotoxin to isolated rat neurohypophysial nerve terminals, its effect on the depolarization-induced increase of cytoplasmic Ca2+ and on the potassium and electrically-induced release of vasopressin (AVP) have been studied. The results show that isolated neurosecretory nerve endings have calcium channels with a high affinity for omega-CgTx and that this toxin inhibits neurohormone release at very low concentration (IC50 = 0. 1nM). Although secretion of vasopressin is inhibited to a great extent by the toxin it is shown that a small but significant amount of the depolarization-induced AVP release is insensitive to omega-CgTx and to the dihydropyridine molecule nicardipine.