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PubMed2004

Regulation of releasable vesicle pool sizes by protein kinase A-dependent phosphorylation of SNAP-25.

Protein kinase A (PKA) is a key regulator of neurosecretion, but the molecular targets remain elusive. We combined pharmacological manipulations of kinase and phosphatase activities with mutational studies on the exocytotic machinery driving fusion of catecholamine-containing vesicles from chromaffin cells. We found that constitutive PKA activity was necessary to maintain a large number of vesicles in the release-ready, so-called primed, state, whereas calcineurin (protein phosphatase 2B) activity antagonized this effect. Overexpression of the SNARE protein SNAP-25a mutated in a PKA phosphorylation site (Thr-138) eliminated the effect of PKA inhibitors on the vesicle priming process. Another, unidentified, PKA target regulated the relative size of two different primed vesicle pools that are distinguished by their release kinetics. Overexpression of the SNAP-25b isoform increased the size of both primed vesicle pools by a factor of two, and mutations in the conserved Thr-138 site had similar effects as in the a isoform.

Alanine↗
PubMed1996

Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: correlation with its blockade of catecholamine release.

The seven types (A--G) of botulinum neurotoxin (BoNT) are Zn2+ -dependent endoproteases that potently block neurosecretion. Syntaxin is presently thought to be the sole substrate for BoNT/C1, and synaptosomal-associated protein of Mr = 25 000 (SNAP-25) is selectively proteolyzed by types A and E. In this study, the effects of C1 on Ca2+ -regulated exocytosis of dense core granules from adreno-chromaffin cells were examined together with its underlying molecular action. Intact chromaffin cells were exposed to the toxin, and catecholamine release therefrom was then measured in conjunction with the monitoring of syntaxin cleavage by Western blotting. A good correlation was obtained between degradation of syntaxin 1A/B and reduction in Ca2+- or Ba2+-dependent secretion. However, blotting with antibodies against a C-terminal peptide of SNAP-25 revealed the additional disappearance of immunoreactivity, with the same toxin concentration dependency as syntaxin breakdown. Notably, the cleaved SNAP-25 product was similar in size to that produced by BoNT/A; however, contamination of BoNT/C1 by serotypes A or E was eliminated. Therefore, it is concluded that syntaxin 1A/B and SNAP-25 are cleaved in intact cells poisoned with only C1. Notably, C1 treatment of chromaffin cells abolished Ca2+ -evoked secretion following digitonin permeabilization, compared with partial inhibition by BoNT/A, suggesting the importance of syntaxin for catecholamine release. Unexpectedly, C1 failed to proteolyze a soluble recombinant SNAP-25, even though it served as an efficient substrate for BoNT/A. These interesting observations suggest that C1 can only efficiently cleave SNAP-25 in intact cells, possibly due to the existence therein of a unique conformation and/or the participation of accessory factors.

Animals↗
PubMed1999

Characterizing the response of calcium signal transducers to generated calcium transients.

Cellular Ca2+ transients and Ca2+-binding proteins regulate physiological phenomena as diverse as muscle contraction, neurosecretion, and cell division. When Ca2+ is rapidly mixed with slow Ca2+ chelators, EGTA, or Mg2+/EDTA, artificial Ca2+ transients (ACTs) of varying duration (0.1-50 ms half-widths (hws)) and amplitude can be generated. We have exposed several Ca2+ indicators, Ca2+-binding proteins, and a Ca2+-dependent enzyme to ACTs of various durations and observed their transient binding of Ca2+, complex formation, and/or activation. A 0.1 ms hw ACT transiently occupied approximately 70% of the N-terminal regulatory sites of troponin C consistent with their rapid Ca2+ on-rate (8.7 +/- 2.0 x 10(7) M-1 s-1). A 1.1 ms hw ACT produced approximately 90% transient binding of the N-terminal of calmodulin (CaM) to the RS-20 peptide, but little binding of CaM's C-terminal to RS-20. A 0.6 ms hw ACT was sufficient for the N-terminal of CaM to transiently bind approximately 60% of myosin light chain kinase (MLCK), while a 1.8 ms hw ACT produced approximately 22% transient activation of the sarcoplasmic reticulum (SR) Ca2+/ATPase. In both cases, the ACT had fallen back to baseline approximately 10-30 ms before maximal binding of CaM to MLCK or SR Ca2+/ATPase activation occurred and binding and enzyme activation persisted long after the Ca transient had subsided. The use of ACTs has allowed us to visualize how the Ca2+-exchange rates of Ca2+-binding proteins dictate their Ca2+-induced conformational changes, Ca2+-induced protein/peptide and protein/protein interactions, and enzyme activation and inactivation, in response to Ca2+ transients of various amplitude and duration. By characterizing the response of these proteins to ACTs, we can predict with greater certainty how they would respond to natural Ca2+ transients to regulate cellular phenomena.

Adenosine Triphosphatases↗
PubMed2001

Neurosecretory granule formation in ligated axons: additional arguments for a local differentiation from a Golgi apparatus extension.

The sorting domain for the different types of granules and small synaptic vesicles in neurosecretion is still largely a matter of debate. Some authors state that an exocytotic process has to precede granule formation. In previous studies, we favoured the idea that neurosecretory packages in terminals are assembled from axonal reticulum membranes simply by differentiation at the axon ending, the axonal reticulum being an extension of the Golgi apparatus. By ligating bovine splenic nerve, a de novo differentiation can be induced. After ligation, granules and granulo-tubular complexes appear. They were immunoreactive for SV2, VMAT2 and synaptobrevin II, which are all known to be highly enriched in large dense granules. Previously the granulo-tubular structures have already been recognized as precursor stadia of neurosecretory granules. It is concluded that at a de novo differentiation, a sorting out and aggregation is taking place of molecules typical for large dense granules. The small dense granules and tubules can be considered unripe, precursor forms of the large dense granules. All this occurs in the absence of signs of exocytosis. The present findings corroborate the view that granule formation occurs via local differentiation at an axon ending.

Animals↗
PubMed1998

Membrane excitability and secretion from peptidergic nerve terminals.

1. Thin slices of the posterior pituitary can be used as a preparation for the study of biophysical mechanisms underlying neuropeptide secretion. Patch-clamp techniques in this preparation have revealed the properties of ion channels that control the excitability of the nerve terminal membrane and have clarified the relation between Ca2+ and exocytosis. 2. Repetitive electrical activity at high frequencies broadens action potentials to allow more Ca2+ entry and thus enhance exocytosis. Action potential broadening results from the inactivation of a voltage-dependent K+ channel. 3. When repetitive electrical activity is sustained, secretion is depressed. This depression can be attributed in part to action potential failure caused by the opening of a Ca(2+)-activated K+ channel. This channel can be modulated by protein kinases, phosphatases, and G-proteins. 4. The inhibitory neurotransmitter GABA activates a GABAA receptor in the nerve terminal membrane. The gating of the associated Cl- channel depolarizes the membrane slightly to inactivate voltage-gated Na+ channels and block action potential propagation. 5. The response of the nerve terminal GABAA receptor is enhanced by neuroactive steroids and this can potentiate the inhibition of neurosecretion by GABA. The action of neurosteroids at this site could play a role in changes in neuropeptide secretion associated with reproductive transitions. 6. Ca2+ channels in the nerve terminal membrane are inactivated by sustained depolarization and by trains of brief pulses. Ca2+ entry promotes Ca2+ channel inactivation during trains by inhibiting the recovery of Ca2+ channels from inactivation. The inactivation of Ca2+ channels can play a role in defining the optimal frequency and train duration for evoking neuropeptide secretion. 7. Measurements of membrane capacitance in peptidergic nerve terminals have revealed rapid exocytosis and endocytosis evoked by Ca2+ entry through voltage-gated Ca2+ channels. Exocytosis is too rapid to account for the delays in neuropeptide secretion evoked by trains of action potentials. Endocytosis sets in rapidly after exocytosis with a time course comparable to that of the rapid endocytosis observed in nerve terminals at rapid synapses. Our results support the finding in rapid synaptic nerve terminals that endocytosis is inhibited by intracellular Ca2+. Multiple pools of vesicles were revealed, and these pools may reflect different stages in the mobilization and release of neuropeptide.

Action Potentials↗
PubMed1998

Synaptic modulation of oscillatory activity of hypothalamic neuronal networks in vitro.

1. Rhythmic bursts of action potentials in neurosecretory cells are a key factor in hypothalamic neurosecretion. Rhythmicity and synchronization may be accomplished by pacemaker cells synaptically driving follower cells or by a network oscillator. 2. In this review we describe a hypothalamic cell culture which may serve as a model for a hypothalamic network oscillator. An overview is given of neurochemical phenotypes, synaptic mechanisms and their development, properties of receptors for fast synaptic transmission, and membrane properties of cells in dissociated rat embryonic hypothalamic culture. 3. Rhythmic activity spreads in the cultured network through synapses that release glutamate, activating a heteromultimeric AMPA-type receptor containing a GluR2 subunit which is associated with a high-conductance channel for Na+ and K+. Rhythmic activity is controlled by synapses that release GABA to activate GABAA receptors. The presumed function of the two receptor types is facilitated by their respective location, GABAA receptors predominating near the soma and AMPA receptors being abundant in dendrites. 4. Network oscillators may be more reliable for the presumed function than single-cell oscillators. They are controlled through synaptic modulation, which may prove to represent a process important for the release of hormones.

Animals↗
PubMed2002

Calcium-dependent transferrin receptor recycling in bovine chromaffin cells.

The release of regulated secretory granules is known to be calcium dependent. To examine the Ca2+-dependence of other exocytic fusion events, transferrin recycling in bovine chromaffin cells was examined. Internalised 125I-transferrin was released constitutively from cells with a half-time of about 7 min. Secretagogues that triggered catecholamine secretion doubled the rate of 125I-transferrin release, the time courses of the two triggered secretory responses being similar. The triggered 125I-transferrin release came from recycling endosomes rather than from sorting endosomes or a triggered secretory vesicle pool. Triggered 125I-transferrin release, like catecholamine secretion from the same cells, was calcium dependent but the affinities for calcium were very different. The extracellular calcium concentrations that gave rise to half-maximal evoked secretion were 0.1 mm for 125I-transferrin and 1.0 mm for catecholamine, and the intracellular concentrations were 0.1 microm and 1 microm, respectively. There was significant 125I-transferrin recycling in the virtual absence of intracellular Ca2+, but the rate increased when Ca2+ was raised above 1 nm, and peaked at 1 microm when the rate had doubled. Botulinum toxin type D blocked both transferrin recycling and catecholamine secretion. These results indicate that a major component of the vesicular transport required for the constitutive recycling of transferrin in quiescent cells is calcium dependent and thus under physiological control, and also that some of the molecular machinery involved in transferrin recycling/fusion processes is shared with that for triggered neurosecretion.

Animals↗
PubMed1980

Cholecystokinin octapeptide in the rat hypothalamo-neurohypophysial system.

Gastrin-cholecystokinin (CCK)-like immunoreactivity has been visualized by immunohistochemistry in the vasopressin-oxytocin neurosecretory system comprising the posterior lobe of the pituitary, the supraoptic and paraventricular nuclei of the hypothalamus. This CCK-like substance has not been chemically identified in the rat, but has been reported to be gastrin 17 in the swine pituitary. We report here that this CCK-like immunoreactive substance co-chromatographs with CCK8 sulphate on Sephadex G-50 and two HPLC chromatographic systems. No gastrin 17 was detected in rat pituitary or brain. We further report that about 60% of the CCK in posterior lobe originates in cell bodies in the paraventricular nucleus of the hypothalamus. The CCK content of posterior pituitary is dramatically decreased by physiological perturbations which stimulate vasopressin or oxytocin release. We propose that CCK may either be co-secreted with vasopressin and oxytocin to act on peripheral targets or may be involved in the regulation of vasopressin or oxytocin neurosecretion.

Animals↗
PubMedDate not supplied

GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels.

The modulation of voltage-dependent calcium channels by hormones and neurotransmitters has important implications for the control of many Ca2+-dependent cellular functions including exocytosis and contractility. We made use of electrophysiological techniques, including whole-cell patch-clamp recordings from dorsal root ganglion (DRG) neurones, to demonstrate a role for GTP-binding proteins (G-proteins) as signal transducers in the noradrenaline- and gamma-aminobutyric acid (GABA)-induced inhibition of voltage-dependent calcium channels. This action of the transmitters was blocked by: (1) preincubation of the cells with pertussis toxin (a bacterial exotoxin catalysing ADP-ribosylation of G-proteins); or (2) intracellular administration of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S), a non-hydrolysable analogue of GDP that competitively inhibits the binding of GTP to G-proteins. Our findings provide the first direct demonstration of the G-protein-mediated inhibition of voltage-dependent calcium channels by neurotransmitters. This mode of transmitter action may explain the ability of noradrenaline and GABA to presynaptically inhibit Ca2+-dependent neurosecretion from DRG sensory neurones.

Action Potentials↗
PubMed1992

MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin.

AGONISTS that stimulate protein kinase C (PKC) induce profound changes in cell morphology correlating with the reorganization of submembranous actin, but no direct connection between PKC and actin assembly has been identified. The myristoylated, alanine-rich C kinase substrate (MARCKS) binds calmodulin and is a predominant, specific substrate of PKC which is phosphorylated during macrophage and neutrophil activation , growth factor-dependent mitogenesis and neurosecretion; it is redistributed from plasma membrane to cytoplasm when phosphorylated and is involved in leukocyte motility. Here we report that MARCKS is a filamentous (F) actin crosslinking protein, with activity that is inhibited by PKC-mediated phosphorylation and by binding to calcium-calmodulin. MARCKS may be a regulated crossbridge between actin and the plasma membrane, and modulation of the actin crosslinking activity of the MARCKS protein by calmodulin and phosphorylation represents a potential convergence of the calcium-calmodulin and PKC signal transduction pathways in the regulation of the actin cytoskeleton.

Actin Cytoskeleton↗
PubMed1997

Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms.

The cystic fibrosis gene encodes a cyclic AMP-gated chloride channel (CFTR) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. The molecular mechanisms that modulate CFTR activity in epithelial tissues are poorly understood. Here we show that CFTR is regulated by an epithelially expressed syntaxin (syntaxin 1A), a membrane protein that also modulates neurosecretion and calcium-channel gating in brain. Syntaxin 1A physically interacts with CFTR chloride channels and regulates CFTR-mediated currents both in Xenopus oocytes and in epithelial cells that normally express these proteins. The physical and functional interactions between syntaxin 1A and CFTR are blocked by a syntaxin-binding protein of the Munc18 protein family (also called n-Secl). Our results indicate that CFTR function in epithelial cells is regulated by an interplay between syntaxin and Munc18 isoforms.

Animals↗
PubMed1994

Residual Ca2+ and short-term synaptic plasticity.

At many synapses, the amount of transmitter released by action potentials increases progressively during a train of spikes. This enhancement of evoked transmitter release grows during tetanic stimulation with several time constants, each bearing a different name (facilitation: tens to hundreds of milliseconds; augmentation: several seconds; potentiation: several minutes), and the enhancement of release to test spikes after a tetanus decays with similar time constants. All these processes depend on presynaptic Ca2+ influx during the conditioning tetanus. It has often been proposed that these forms of synaptic plasticity are due to residual Ca2+ present in nerve terminals following conditioning activity. We tested this idea directly by using photolabile Ca2+ chelators to reduce residual Ca2+ following conditioning stimulation or to generate an artificial elevation in Ca2+ concentration, and observed the effects on synaptic transmission at crayfish neuromuscular junctions. We found that facilitation, augmentation and potentiation are caused by the continuing action of residual Ca2+. Augmentation and potentiation seem to arise from Ca2+ acting at a separate site from facilitation, and these sites are different from the molecular target triggering neurosecretion.

Action Potentials↗
PubMed1999

Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel.

Voltage-gated ion channels underlie the generation of action potentials and trigger neurosecretion and muscle contraction. These channels consist of an inner pore-forming domain, which contains the ion permeation pathway and elements of its gates, together with four voltage-sensing domains, which regulate the gates. To understand the mechanism of voltage sensing it is necessary to define the structure and motion of the S4 segment, the portion of each voltage-sensing domain that moves charged residues across the membrane in response to voltage change. We have addressed this problem by using fluorescence resonance energy transfer as a spectroscopic ruler to determine distances between S4s in the Shaker K+ channel in different gating states. Here we provide evidence consistent with S4 being a tilted helix that twists during activation. We propose that helical twist contributes to the movement of charged side chains across the membrane electric field and that it is involved in coupling voltage sensing to gating.

Animals↗
PubMed2001

The proteins of synaptic vesicle membranes are affected during ageing of rat brain.

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.

Aging↗
PubMed2002

Phosphorylation of 46-kDa protein of synaptic vesicle membranes is stimulated by GTP and Ca2+/calmodulin.

The release of neurotransmitter is regulated in the processes of membrane docking and membrane fusion between synaptic vesicles and presynaptic plasma membranes. Synaptic vesicles contain a diverse set of proteins that participate in these processes. Small GTP-binding proteins exist in the synaptic vesicles and are suggested to play roles for the regulation of neurotransmitter release. We have examined a possible role of GTP-binding proteins in the regulation of protein phosphorylation in the synaptic vesicles. GTPgammaS stimulated the phosphorylation of 46 kDa protein (p46) with pI value of 5.0-5.2, but GDPbetaS did not. The p46 was identified as protein interacting with C-kinase 1 (PICK-1) by MALDI-TOF mass spectroscopy analysis, and anti-PICK-1 antibody recognized the p46 spot on 2-dimensional gel electrophoresis. Rab guanine nucleotide dissociation inhibitor (RabGDI), which dissociates Rab proteins from SVs, did not affect phosphorylation of p46. Ca(2+)/calmodulin (CaM), which causes the small GTP-binding proteins like Rab3A and RalA to dissociate from the membranes and stimulates CaM-dependent protein kinase(s) and phosphatase, strongly stimulate the phosphorylation of p46 in the presence of cyclosporin A and cyclophylin. However, RhoGDI, which dissociates Rho proteins from membranes, reduced the phosphorylation of p46 to the extent of about 50%. These results support that p46 was PICK-1, and its phosphorylation was stimulated by GTP and Ca(2+)/CaM directly or indirectly through GTP-binding protein(s) and Ca(2+)/CaM effector protein(s). The phosphorylation of p46 (PICK-1) by GTP and Ca(2+)/CaM may be important for the regulation of transporters and neurosecretion.

Animals↗
PubMed1983

The neurohypophysial axons in normal and dehydrated rats: an electron microscopic study including ZIO-staining method.

The neurohypophysial axons from normal rats and those dehydrated for 4 and 12 days were investigated by means of conventional glutaraldehyde fixation and the zinc-iodide-osmium (ZIO) staining technique. Conventionally fixed axons from dehydrated rats revealed depletion of dense core of neurosecretory granules, more frequent occurrence of microvesicles, proliferation of smooth endoplasmic reticulum or accumulation of lysosomes. It has been disclosed that the ZIO reaction product has the ability to stain microvesicles (normal rats) and smooth endoplasmic reticulum, lysosomes, some neurosecretory granules and some parts of stromal inclusion at the release site (dehydrated rats). Quantitative data are also presented. They indicate a significantly decreased number of both neurosecretory granules (NSG) and ZIO-positive microvesicles (MV) in dehydrated rats. Equally, the value of the median of the MV/NSG ratio was significantly increased from 1.38 in normal rats to 5.50 and 7.50 in rats dehydrated for 4 and 12 days, respectively. Thus, the possible involvement of ZIO-positive microvesicles in a process associated with neurosecretion is suggested. While in normal rats a correlation was found between the number of neurosecretory granules and the area of sectioned profiles of the axons, no evidence was obtained for it in 12-day dehydrated rats. Conversely, no evidence for a relationship between the axon area and the number of ZIO-positive microvesicles was obtained in normal rats, while in rats dehydrated for 12 days a correlation was found.

Animals↗
PubMed2002

Allelic association of the neuronal nitric oxide synthase (NOS1) gene with schizophrenia.

Nitric oxide (NO) has been identified as a widespread and multifunctional biological messenger molecule in the central nervous system (CNS), with possible roles in neurotransmission, neurosecretion, synaptic plasticity, and tissue injury in many neurological disorders, including schizophrenia. Neuronal NO is widely produced in the brain from L-arginine catalyzed by neuronal NO synthase (NOS1). We therefore hypothesized that the NOS1 gene may play a role in the pathophysiology of schizophrenia. In the present study, we examined the genetic association between a novel single nucleotide polymorphism (SNP: a C-->T transition located 276 base pairs downstream from the translation termination site) of the human NOS1 gene, which is located in chromosome 12q24, and schizophrenia (215 Japanese patients with schizophrenia and 182 healthy controls). The allele frequencies of the polymorphism in exon 29 of the NOS1 gene differed significantly between patients with schizophrenia and controls (chi(2) = 20.10, df = 1, P = 0.000007; relative risk = 1.92; 95% confidence interval = 1.44-2.55). Our results suggest that the NOS1 gene polymorphism may confer increased susceptibility to schizophrenia.

Alleles↗
PubMed2002

Cross-talk unfolded: MARCKS proteins.

The proteins of the MARCKS (myristoylated alanine-rich C kinase substrate) family were first identified as prominent substrates of protein kinase C (PKC). Since then, these proteins have been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as endo-, exo- and phago-cytosis, and neurosecretion. The effector domain of MARCKS proteins is phosphorylated by PKC, binds to calmodulin and contributes to membrane binding. This multitude of mutually exclusive interactions allows cross-talk between the signal transduction pathways involving PKC and calmodulin. This review focuses on recent, mostly biophysical and biochemical results renewing interest in this protein family. MARCKS membrane binding is now understood at the molecular level. From a structural point of view, there is a consensus emerging that MARCKS proteins are "natively unfolded". Interestingly, domains similar to the effector domain have been discovered in other proteins. Furthermore, since the effector domain enhances the polymerization of actin in vitro, MARCKS proteins have been proposed to mediate regulation of the actin cytoskeleton. However, the recent observations that MARCKS might serve to sequester phosphatidylinositol 4,5-bisphosphate in the plasma membrane of unstimulated cells suggest an alternative model for the control of the actin cytoskeleton. While myristoylation is classically considered to be a co-translational, irreversible event, new reports on MARCKS proteins suggest a more dynamic picture of this protein modification. Finally, studies with mice lacking MARCKS proteins have investigated the functions of these proteins during embryonic development in the intact organism.

Amino Acid Sequence↗