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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 int racellular Ca2+concentration ([Ca2+]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 μM. The magnitude order for binding of different nucleotides was α β-Met-ATP ≥ dATPαS > ATP ≥ 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 μM) caused a concentration-dependent increase in [Ca2+]i(EC50= 30 μM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca2+]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 Ca2+entry and increase of [Ca2+]i. ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca2+]e. Similarly, α,β-Met-ATP, a potent P2x agonist also stimulated Ca2+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 Ca2+entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is alsoable to induce actin disaggregation by a Ca2+dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis.

1. Isolated neurosecretory nerve endings were prepared from rat neurohypophyses. The amount of vasopressin (AVP) and oxytocin released was measured by radioimmunoassay. 2. The amount of hormone release under resting conditions was not affected by external calcium (Ca2+o). Secretion decreased by ca. 50% when external sodium (Na+o) was replaced by choline or sucrose. 3. Ouabain did not modify the basal AVP release. 4. The Na+ ionophore monensin increased the release of AVP only in the presence of Na+o. This increase was maintained during prolonged exposure to the ionophore and occurred in the presence of Ca2+o only. 5. In the presence of Ca2+o, the amount of evoked hormone release was dependent on the external K+ concentration. Half‐maximal activation was achieved with ca. 40 mM‐K+. The K+‐induced secretion was potentiated in Na+‐free solution. 6. Prolonged 100 mM‐K+‐induced depolarization in the presence of Ca2+o gave rise to a large increase in hormone secretion which decreased with time (t1/2 = 2.5 min). The release could be reactivated after permeabilization of the nerve terminals in the presence of micromolar concentrations of Ca2+. 7. A stepwise paradigm in which Ko+ is incrementally increased to 25, 50, 75 and then 100 mM released more AVP than a prolonged exposure to 100 mM‐K+. 8. Veratridine increased the amount of AVP released. This effect was considerably reduced in the absence of Nao+ and abolished in the presence of D600. 9. The depolarization‐induced AVP release was blocked by different Ca2+‐antagonists. Their effectiveness was nitrendipine = nicardipine greater than Cd2+ greater than Gd3+ greater than Co2+ = Mn2+. 10. The dihydropyridine Bay K 8644 potentiated both the basal and the K+‐evoked AVP release. Its maximal effect was obtained with 25‐50 mM‐Ko+. 11. In conclusion, the isolated neurohypophysial terminals which have both Na+ and Ca2+ channels and release AVP and oxytocin upon depolarization might be an excellent system to study further the mechanisms leading to secretion of neurohormones.

1. Isolated nerve endings from rat neurohypophyses were permeabilized with digitonin in order to gain access to the cytoplasm. Release of vasopressin (AVP), oxytocin and the neurophysins was studied under different experimental conditions. 2. Hormone release, which occurred by exocytosis, was Ca2+ dependent. Half‐maximal release was observed at ca. 1.7 microM‐Ca2+ in contrast to ca. 300 microM for K+‐induced hormone secretion from non‐permeabilized neurosecretosomes. 3. Release also occurred when the neurosecretosomes were challenged with Ca2+ 20 min after digitonin treatment. This suggests that the isolated nerve endings remain permeable after treatment with digitonin. 4. Although hormone release was potentiated in the presence of ATP, and to a lesser extent with guanosine triphosphate (GTP), secretion occurred in the absence of nucleotides. 5. Replacement of K+ as the major cation by Na+ did not modify the secretory response to a Ca2+ challenge. Release, although reduced, still occurred when KCl was replaced by sucrose. 6. Compared to glutamate, Cl‐, Br‐ and I‐ did not modify the Ca2+‐independent release. This release was increased in the presence of SCN‐. The order of effectiveness of the anions studied in inhibiting the Ca2+‐dependent release was glutamate less than Br‐ = Cl‐ = I‐ less than SCN‐. 7. Increasing the osmolarity of the perfusate inhibited the Ca2+‐dependent release of AVP and oxytocin. 8. Vincristine, which binds to microtubules, had no effect on the secretory process. 9. Ca2+ dependent AVP release was partially inhibited by the calmodulin antagonist trifluoroperazine. 10. Hormone release was potentiated by the protein kinase C activator, 4‐beta‐phorbol 12‐myristate acetate (TPA). 11. Whereas 0.2 microM‐Ca2+ induced a barely significant increase in AVP release, inositol 1,4,5‐triphosphate, in the continued presence of 0.2 microM‐Ca2+, produced a large secretory response. 12. 4‐acetamido‐4'‐isothiocyanostilbene‐2,2'‐disulphonic acid (SITS), an inhibitor of Cl‐ permeability, reduced the Ca2+‐dependent AVP release. 13. Carbonyl cyanide m‐chlorophenylhydrazone (CCCP), which reduces the transmembrane potential of isolated neurohypophysial granules, inhibited the Ca2+‐dependent hormone secretion. 14. Maximal hormone release occurred at pH 6.6. 15. It is concluded that the permeabilized neurosecretosomes represent an excellent model for studying the minimal requirements for neurosecretion.

Rat neurointermediate lobes and neurohypophyses separated from the pars intermedia were stimulated in vitro in the presence of either D-Ala2, D-Leu5-enkephalin (DADLE), a Leu-enkephalin stable analogue or FK 33-824 a Met-enkephalin stable analogue. Secretion of vasopressin (AVP) and oxytocin (OT) was produced by either a Ca2+-ionophore or with electrical stimulation or by K+-induced depolarization. These opioid peptides and their antagonist naloxone did not affect basal nor evoked hormone release. Furthermore, they did not affect the evoked calcium uptake induced with electrical stimulation. These findings were confirmed using a preparation of isolated neurosecretory nerve endings. Further, dopamine had no effect on the K+-induced AVP release although a crude extract of the pars intermedia abolished the electrically-evoked and reduced considerably the potassium-evoked AVP release. It is concluded that in the neurohypophysis neither Leu- and Met-enkephalin nor dopamine affect the secretion-coupling mechanism at the level of the neurosecretory nerve endings.

Isolated rat neural lobes were stimulated electrically and the release of vasopressin and oxytocin was measured by radioimmunoassay. The neurohypophyses were stimulated with pulses given at a constant frequency or with a pulse pattern imitating the electrical activity, recorded in vivo, of vasopressin‐ or oxytocin‐containing magnocellular neurones. A single burst recorded from a 'vasopressin' cell with an intraburst mean frequency of 13 Hz evoked more vasopressin release than the same number of stimuli delivered at a constant frequency of 13 Hz. The amount of vasopressin release per pulse was much higher at the beginning than at the end of the burst. Series of bursts given with interburst silent periods released more hormone than bursts delivered without silent periods. The amount of hormone released by four 'vasopressin' bursts was significantly larger with silent periods of 21 s than with shorter intervals. Four pulses were much more effective in promoting hormone release when given with 60 ms interspike intervals at the beginning of each second than when delivered at a constant frequency of 4 Hz. Prolonged stimulation with 'vasopressin' bursts had a greater effect in inducing hormone release than the same number of pulses given in burst delivered at a constant frequency of 13 Hz. After an initial increase the rate of vasopressin release declined rapidly whereas oxytocin release remained elevated for the first 20 min and only then decreased. The release of both vasopressin and oxytocin remained, however, above the release from unstimulated neurohypophyses. 45Ca uptake in the neural lobe was larger when the neurohypophyses were stimulated with vasopressin or oxytocin bursts delivered with silent intervals than when the silent periods were omitted, or when the tissue was stimulated with bursts with the same number of pulses but given at a constant frequency of 13 Hz. In conclusion, it is suggested that the interspike intervals in a burst and the silent intervals between bursts are two important determinants of the effectiveness of the burst pattern in promoting neuropeptide release.

We have investigated the role of endothelin (ET) in the stimulus-secretion coupling mechanism in the posterior pituitary. We report that isolated nerve endings contain immunoreactive endothelin, the level of which is regulated by homeostatic mechanisms involved in control of water balance. ET-1 and ET-3 potentiate vasopressin release induced by depolarization through interaction with specific receptors of the ETA subtype and this response is antagonized by sarafotoxin S6b. The second messenger for this effect, however, remains unknown since the potentiation of depolarization-induced vasopressin release occurs in the absence of an increase in cellular calcium.

The aim of the present study was to investigate how atriopeptin inhibits secretagogue-stimulated ACTH secretion in vitro. Perifused isolated rat anterior pituitary cells were used throughout; the ACTH content of the perifusate was measured by radioimmunoassy.In the presence of a constant (0·05 nmol/l) concentration of 41-residue corticotrophin-releasing factor (CRF), arginine vasopressin (AVP; 0·05–50 nmol/l) stimulated ACTH secretion in a concentration-dependent manner, the combination of 0·05 nmol CRF/l and 0·5 nmol AVP/l (CRF/AVP) stimulated ACTH release to six- to eightfold above baseline. The effect of CRF/AVP was not modified by tetrodotoxin, but was abolished by CoCl2and reduced to about 70% of the control stimulus by nifedipine.Application of 103–126 residue atriopeptin for 10 min before and 2·5 min during the CRF/AVP stimulus strongly suppressed the evoked release of ACTH, the maximal inhibition was 75–90% at 10 nmol atriopeptin/l. The calcium ionophore ionomycin (200 nmol/l) reversed the effect of atriopeptin while it had no secretagogue activity of its own, and did not enhance the response to CRF/AVP. A variety of blockers of K+channels, 4-amino pyridine, tetraethylammonium, apamine, quinine, but not tolbutamide, effectively antagonized the inhibitory action of atriopeptin (10 nmol/l). None of these drugs altered ACTH release evoked by CRF/AVP. In concentration–response experiments, the half effective concentration of 4-aminopyridine and tetraethyl-ammonium were around 1 mmol/l and 10 nmol/l for apamine. Finally, tetraethylammonium and apamine also antagonized the inhibition of CRF/AVP-evoked ACTH release by 8-Br-cGMP.These data suggest that (1) at least two types of K+channels, a delayed rectifier and the apamine-sensitive Ca2+-activated channel, are functionally important in pituitary corticotroph cells; (2) atriopeptin inhibits CRF/AVP-stimulated ACTH secretion by hyperpolarizing the plasma membrane and thus reducing the uptake of Ca2+into the cells; (3) cGMP is the intracellular mediator of the action of atriopeptin on corticotroph cells.Journal of Endocrinology(1990) 126,183–191

The aim of the present study was to characterize the inhibitory action of atriopeptin on secretagogue-evoked ACTH release in vitro.Perifused isolated rat anterior pituitary cells were exposed to repeated pulses of 41-residue corticotrophin-releasing factor (CRF-41) or arginine vasopressin (AVP). The net ACTH secretory response to both neurohormones increased progressively with the number of pulses applied, until a maximum hormonal response was reached which was stable for the subsequent period of observation (2–3 h). The maximal secretagogue-evoked hormone release eventually achieved was 4 and 1·7 times greater than the initial response to AVP and CRF-41 respectively. The size of the ACTH response elicited by 50 pmol CRF-41/1 and 500 pmol AVP/1 (CRF/AVP) given together also underwent progressive enhancement. The number of secretagogue pulses required to reach the maximal response to a particular stimulus depended upon the concentration of the secretagogue peptides, higher concentrations favoured a more rapid development of the stable secretory response.The potency of 103–126 residue atriopeptin to inhibit CRF/AVP-induced ACTH release varied by about 1000-fold depending upon the prior treatment of the cells. In general, cells not previously exposed to secretagogues appeared largely resistant, those under a moderate secretagogue drive were strongly inhibited, and those under intense stimulation were again refractory to inhibition by atriopeptin. In contrast, corticosterone suppressed stimulated ACTH release regardless of the state of the cells.The data demonstrate that the conditions of cell maintenance are pivotal determinants of the inhibitory effect of atriopeptin on secretagogue-stimulated ACTH release in vitro. The in-vivo correlate of these findings may be that the sensitivity of corticotrophs towards atriopeptin is determined by requirements encoded through the pattern of release of hypothalamic CRF-41 and AVP.Journal of Endocrinology(1990) 125,365–373

The current concept of the regulation of adenohypophysial corticotrophin secretion assigns a stimulatory role to neuropeptides of hypothalamic origin and an inhibitory function to adrenocortical glucocorticoid hormones. It has been shown that in isolated perifused rat anterior pituitary cells atriopeptins (1–28) and (5–28), but not (5–25), potently suppress corticotrophin secretion elicited by 41-residue corticotrophin-releasing factor and arginine vasopressin. The action of atriopeptins is associated with a marked increase in the cellular content of cyclic GMP. Moreover, 8-Br-cyclic GMP, a cell membrane permeant analogue of cyclic GMP, also inhibited secretagogue-stimulated corticotrophin release. It is suggested that atriopeptins of hypothalamic and perhaps of cardiac origin may be functionally significant corticotrophin-release inhibiting factors.Journal of Endocrinology(1990) 125,39–44

THE influence of calcium currents expressed at early stages on the subsequent development of calcium currents was studied in embryonic rat hypothalamic neurones in culture. Voltage-activated calcium currents and spontaneous fluctuations of intracellular free calcium concentration ([Ca2+]i) were monitored. Acute application of nickel chloride (0.1 mM) to 6− to 7-day-old cultures strongly reduced calcium currents and [Ca2+]ifluctuations. When cultures were maintained for 6–7 days in the presence of NiCl2(0.05–0.1 mM), expression of the low voltage-activated current was strongly inhibited; this treatment did not affect high voltage-activated currents. Our results suggest that spontaneous activation of calcium currents early during development promotes calcium influx that regulates expression of calcium current in mature neurones.

1. Intracellular Ca2+ concentration ([Ca2+]i) was monitored in single cells isolated from adult rat supraoptic (SO) nuclei. The great majority of cells (85%) were neurones and most were immunoreactive to oxytocin or to vasopressin (AVP). 2. The resting [Ca2+]i of the majority (80%) of the neurones remained stable while 20% of the neurones displayed spontaneous [Ca2+]i oscillations which disappeared in low‐Ca2+ (100 nM) EGTA buffer. 3. Addition of 100 nM oxytocin increased the [Ca2+]i in both stable and oscillating cells. Two types of responses were observed: (i) a sustained response with [Ca2+]i being maintained at an elevated level and (ii) a brief response with [Ca2+]i quickly returning to a near‐resting level. Responses were reproducible, dose dependent and blocked with a specific oxytocin antagonist. 4. Removal of extracellular Ca2+ did not block the oxytocin response. In EGTA buffer, application of thapsigargin (200 nM) onto oxytocin‐sensitive cells induced an increase in [Ca2+]i and inhibited the oxytocin response. These effects were not induced by other intracellular Ca2+ mobilizers such as tBuBHQ (see Methods) or caffeine. 5. In conclusion, half of the SO cells respond to oxytocin with a rise in [Ca2+]i. The effect is mediated by oxytocin receptors and results from release of Ca2+ from thapsigargin‐sensitive stores.

1. The intracellular Ca2+ concentration ([Ca2+]1) was monitored in single magnocellular neurones freshly isolated from rat supraoptic nucleus. Application of 100 nM vasopressin increased [Ca2+]1. Two types of [Ca2+]1 responses were observed: (i) a transient response, displayed by 86% of the vasopressin‐sensitive neurones, and (ii) a sustained response displayed by 14% of the vasopressin‐sensitive neurones. 2. Among responding neurones, 52% were vasopressin sensitive, 44% were oxytocin sensitive and 4% were sensitive to both peptides. 3. Responses to vasopressin were dose dependent, showed a progressive desensitization after successive applications, were specifically blocked by the V1a vasopressin receptor antagonist, SR 49059, and were unaffected by the oxytocin receptor antagonist, d(CH2)5OVT. 4. Vasopressin responses were completely suppressed by the removal of external Ca2+. 5. The intracellular Ca2+ mobilizers, caffeine and tBuBHQ, did not affect resting or vasopressin‐induced [Ca2+]1 changes. Thapsigargin (200 nM) on its own evoked an increase in [Ca2+]1, and reduced the [Ca2+]1 increase evoked by vasopressin by 52%, suggesting that thapsigargin‐sensitive Ca2+ stores are partially involved in the vasopressin response. 6. Immunocytochemical identification revealed that vasopressin‐responding neurones synthesize vasopressin whereas oxytocin‐responding neurones synthesize oxytocin. 7. In conclusion, vasopressin‐ (partially external Ca2+ dependent) and oxytocin (totally external Ca2+ independent)‐induced [Ca2+]1 changes are mediated by specific receptors. In addition, vasopressin and oxytocin neurones are specifically autoregulated by their own peptides.

The free Ca2+ concentration in isolated rat neurohypophysial nerve endings was measured using the Ca2+ indicator fura-2. Depolarization with high K, veratridine, or electrical stimulation induced an increase in intracellular Ca2+ concentration that was abolished by agents known to block voltage-sensitive Ca channels. Electrical stimulation of the isolated nerve endings with a pulse pattern similar to that recorded in vivo from the hypothalamic magnocellular neurons showed that the increase in intracellular Ca2+ concentration was not only a function of the applied frequency but also of the duration of the silent interburst intervals. The relationship between the cytoplasmic free Ca concentration and the release of neuropeptides is discussed.

The effects of EtOH on peptide release and on high-threshold, voltage-activated calcium (Ca++) channels were examined in acutely dissociated rat neurohypophysial terminals. These terminals release the peptide hormones, arginine vasopressin (AVP) and oxytocin. Release of AVP from isolated intact neurohypophyses, induced by either electrical stimulation or elevated potassium, was inhibited by clinically relevant concentrations of EtOH. "Whole-cell" patch-clamp recording methods were used to study the effects of EtOH on voltage-activated Ca++ currents (ICa) in the peptidergic nerve terminals. Amplitudes of both fast-inactivating ICa and long-lasting ICa were reduced in EtOH, and the reduction in ICa did not result from a shift in its current-voltage or steady-state inactivation relationships. Only the fast-inactivating component recovered after removal of EtOH. The effects of EtOH on ICa could not be attributed to changes in osmolarity. In contrast to ICa, the fast, transient K+ current was insensitive to EtOH. These results suggest that EtOH-induced reduction of ICa in the peptidergic nerve terminals produces a decrease in AVP release, resulting in lowered plasma AVP levels.