An R-type Ca(2+) 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

Ethanol directly modulates gating of a dihydropyridine-sensitive Ca2+ channel in neurohypophysial terminals

Ingestion of ethanol results in a decreased level of plasma vasopressin, which appears to be caused by inhibition of arginine vasopressin (AVP) release from the neurohypophysis. Activation of membrane voltage-gated Ca2+ channels plays an important role in triggering this neurohormone release. In this article, single-channel recordings are used to demonstrate that ethanol, at concentrations constituting legal intoxication, inhibits dihydropyridine-sensitive L-type Ca2+ channels in isolated nerve terminals of the rat neurohypophysis. Ethanol reduced the channel open probability in a concentration-dependent manner. To allow finer resolution of channel openings and to better characterize the mechanisms of ethanol action, Bay K 8644 was used to prolong the openings of L-type Ca2+ channels. In the presence of this dihydropyridine (DHP), the reduction of the channel open probability by concentrations of ethanol of 25 mM or higher could be determined to be due primarily, although not completely, to a shortening of the open duration of this L-channel. Channel conductance was unaffected by ethanol, even at high concentrations. These results are consistent with previous macroscopic data indicating that calcium channels in these peptidergic terminals are targets for ethanol action, and indicate that ethanol acts directly on the gating characteristics of the L-type channel. Furthermore, examination of open and closed state transitions, as well as Hill plot analysis, suggests that ethanol\u27s effects on gating are consistent with the interaction of a single drug molecule with a single target site, possibly the L-channel itself

Individual calcium syntillas do not trigger spontaneous exocytosis from nerve terminals of the neurohypophysis

Recently, highly localized Ca(2+) release events, similar to Ca(2+) sparks in muscle, have been observed in neuronal preparations. Specifically, in murine neurohypophysial terminals (NHT), these events, termed Ca(2+) syntillas, emanate from a ryanodine-sensitive intracellular Ca(2+) pool and increase in frequency with depolarization in the absence of Ca(2+) influx. Despite such knowledge of the nature of these Ca(2+) release events, their physiological role in this system has yet to be defined. Such localized Ca(2+) release events, if they occur in the precise location of the final exocytotic event(s), may directly trigger exocytosis. However, directly addressing this hypothesis has not been possible, since no method capable of visualizing individual release events in these CNS terminals has been available. Here, we have adapted an amperometric method for studying vesicle fusion to this system which relies on loading the secretory granules with the false transmitter dopamine, thus allowing, for the first time, the recording of individual exocytotic events from peptidergic NHT. Simultaneous use of this technique along with high-speed Ca(2+) imaging has enabled us to establish that spontaneous neuropeptide release and Ca(2+) syntillas do not display any observable temporal or spatial correlation, confirming similar findings in chromaffin cells. Although these results indicate that syntillas do not play a direct role in eliciting spontaneous release, they do not rule out indirect modulatory effects of syntillas on secretion

mu-Opioid inhibition of Ca2+ currents and secretion in isolated terminals of the neurohypophysis occurs via ryanodine-sensitive Ca2+ stores

mu-Opioid agonists have no effect on calcium currents (I(Ca)) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, mu-opioid receptor (MOR)-mediated inhibition of I(Ca) is reliably demonstrated using the perforated-patch configuration. This suggests that the MOR-signaling pathway is sensitive to intraterminal dialysis and is therefore mediated by a readily diffusible second messenger. Using the perforated patch-clamp technique and ratio-calcium-imaging methods, we describe a diffusible second messenger pathway stimulated by the MOR that inhibits voltage-gated calcium channels in isolated terminals from the rat neurohypophysis (NH). Our results show a rise in basal intracellular calcium ([Ca(2+)]i) in response to application of [D-Ala(2)-N-Me-Phe(4),Gly5-ol]-Enkephalin (DAMGO), a MOR agonist, that is blocked by D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a MOR antagonist. Buffering DAMGO-induced changes in [Ca(2+)]i with BAPTA-AM completely blocked the inhibition of both I(Ca) and high-K(+)-induced rises in [Ca(2+)]i due to MOR activation, but had no effect on kappa-opioid receptor (KOR)-mediated inhibition. Given the presence of ryanodine-sensitive stores in isolated terminals, we tested 8-bromo-cyclic adenosine diphosphate ribose (8Br-cADPr), a competitive inhibitor of cyclic ADP-ribose (cADPr) signaling that partially relieves DAMGO inhibition of I(Ca) and completely relieves MOR-mediated inhibition of high-K(+)-induced and DAMGO-induced rises in [Ca(2+)]i. Furthermore, antagonist concentrations of ryanodine completely blocked MOR-induced increases in [Ca(2+)]i and inhibition of I(Ca) and high-K(+)-induced rises in [Ca(2+)]i while not affecting KOR-mediated inhibition. Antagonist concentrations of ryanodine also blocked MOR-mediated inhibition of electrically-evoked increases in capacitance. These results strongly suggest that a key diffusible second messenger mediating the MOR-signaling pathway in NH terminals is [Ca(2+)]i released by cADPr from ryanodine-sensitive stores

Ca2+ syntillas, miniature Ca2+ release events in terminals of hypothalamic neurons, are increased in frequency by depolarization in the absence of Ca2+ influx

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of approximately 250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of -80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals

Dihydropyridine receptors and type 1 ryanodine receptors constitute the molecular machinery for voltage-induced Ca2+ release in nerve terminals

Ca2+ stores were studied in a preparation of freshly dissociated terminals from hypothalamic magnocellular neurons. Depolarization from a holding level of -80 mV in the absence of extracellular Ca2+ elicited Ca2+ release from intraterminal stores, a ryanodine-sensitive process designated as voltage-induced Ca2+ release (VICaR). The release took one of two forms: an increase in the frequency but not the quantal size of Ca2+ syntillas, which are brief, focal Ca2+ transients, or an increase in global [Ca2+]. The present study provides evidence that the sensors of membrane potential for VICaR are dihydropyridine receptors (DHPRs). First, over the range of -80 to -60 mV, in which there was no detectable voltage-gated inward Ca2+ current, syntilla frequency was increased e-fold per 8.4 mV of depolarization, a value consistent with the voltage sensitivity of DHPR-mediated VICaR in skeletal muscle. Second, VICaR was blocked by the dihydropyridine antagonist nifedipine, which immobilizes the gating charge of DHPRs but not by Cd2+ or FPL 64176 (methyl 2,5 dimethyl-4[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate), a non-dihydropyridine agonist specific for L-type Ca2+ channels, having no effect on gating charge movement. At 0 mV, the IC50 for nifedipine blockade of VICaR in the form of syntillas was 214 nM in the absence of extracellular Ca2+. Third, type 1 ryanodine receptors, the type to which DHPRs are coupled in skeletal muscle, were detected immunohistochemically at the plasma membrane of the terminals. VICaR may constitute a new link between neuronal activity, as signaled by depolarization, and a rise in intraterminal Ca2+

Goal-directed fluid management based on pulse pressure variation monitoring during high-risk surgery: a pilot randomized controlled trial

Abstract\ud \ud \ud \ud Introduction\ud \ud Several studies have shown that maximizing stroke volume (or increasing it until a plateau is reached) by volume loading during high-risk surgery may improve post-operative outcome. This goal could be achieved simply by minimizing the variation in arterial pulse pressure (ΔPP) induced by mechanical ventilation. We tested this hypothesis in a prospective, randomized, single-centre study. The primary endpoint was the length of postoperative stay in hospital.\ud \ud \ud \ud Methods\ud \ud Thirty-three patients undergoing high-risk surgery were randomized either to a control group (group C, n = 16) or to an intervention group (group I, n = 17). In group I, ΔPP was continuously monitored during surgery by a multiparameter bedside monitor and minimized to 10% or less by volume loading.\ud \ud \ud \ud Results\ud \ud Both groups were comparable in terms of demographic data, American Society of Anesthesiology score, type, and duration of surgery. During surgery, group I received more fluid than group C (4,618 ± 1,557 versus 1,694 ± 705 ml (mean ± SD), P < 0.0001), and ΔPP decreased from 22 ± 75 to 9 ± 1% (P < 0.05) in group I. The median duration of postoperative stay in hospital (7 versus 17 days, P < 0.01) was lower in group I than in group C. The number of postoperative complications per patient (1.4 ± 2.1 versus 3.9 ± 2.8, P < 0.05), as well as the median duration of mechanical ventilation (1 versus 5 days, P < 0.05) and stay in the intensive care unit (3 versus 9 days, P < 0.01) was also lower in group I.\ud \ud \ud \ud Conclusion\ud \ud Monitoring and minimizing ΔPP by volume loading during high-risk surgery improves postoperative outcome and decreases the length of stay in hospital.\ud \ud \ud \ud Trial registration\ud \ud NCT00479011The authors thank Maria De Amorim (Paris, France) and Julia Fukushima (São Paulo, SP, Brazil) for help in data analysis, Dr Julia Wendon (London, UK) for reviewing the manuscript, and Dixtal (Sao Paulo, SP, Brazil) for providing the software for the automatic calculation of ?PP.The authors thank Maria De Amorim (Paris, France) and Julia Fukushima (São Paulo, SP, Brazil) for help in data analysis, Dr Julia Wendon (London, UK) for reviewing the manuscript, and Dixtal (Sao Paulo, SP, Brazil) for providing the software for the automatic calculation of ?PP