Biomedical subjects

E P Seward

Publications and source records attributed to E P Seward.

15 recordsLinked to original sources

PubMed2002

Pathways that control cortical F-actin dynamics during secretion.

Chromaffin cells possess a mesh of filamentous actin underneath the plasma membrane which acts as a barrier to the chromaffin vesicles access to exocytotic sites. Disassembly of cortical F-actin in response to stimulation allows the movement of vesicles from the reserve pool to the release-ready vesicle pool and, therefore, to exocytotic sites. The dynamics of cortical F-actin is controlled by two mechanisms: a) stimulation-induced Ca2+ entry and scinderin activation and b) protein kinase C (PKC) activation and MARCKS phosphorylation as demonstrated here by experiments with recombinant proteins, antisense olygodeoxynucleotides and vector mediated transient expressions. Under physiological conditions (i.e., cholinergic receptor stimulation followed by Ca2+ entry), mechanism (a) is the most important for the control of cortical F-actin network whereas when Ca2+ is released from intracellular stores (i.e., histamine stimulation) cortical F-actin is regulated mainly by mechanism b.

Actins↗

PubMed2000

Bidirectional modulation of exocytosis by angiotensin II involves multiple G-protein-regulated transduction pathways in chromaffin cells.

Angiotensin II (AngII) receptors couple to a multitude of different types of G-proteins resulting in activation of numerous signaling pathways. In this study we examined the consequences of this promiscuous G-protein coupling on secretion. Chromaffin cells were voltage-clamped at -80 mV in perforated-patch configuration, and Ca(2+)-dependent exocytosis was evoked with brief voltage steps to +20 mV. Vesicle fusion was monitored by changes in membrane capacitance (DeltaC(m)), and released catecholamine was detected with single-cell amperometry. Ca(2+) signaling was studied by recording voltage-dependent Ca(2+) currents (I(Ca)) and by measuring intracellular Ca(2+) ([Ca(2+)](i)) with fura-2 AM. AngII inhibited I(Ca) (IC(50) = 0.3 nm) in a voltage-dependent, pertussis toxin (PTX)-sensitive manner consistent with G(i/o)-protein coupling to Ca(2+) channels. DeltaC(m) was modulated bi-directionally; subnanomolar AngII inhibited depolarization-evoked exocytosis, whereas higher concentrations, in spite of I(Ca) inhibition, potentiated DeltaC(m) fivefold (EC(50) = 3.4 nm). Potentiation of exocytosis by AngII involved activation of phospholipase C (PLC) and Ca(2+) mobilization from internal stores. PTX treatment did not affect AngII-dependent Ca(2+) mobilization or facilitation of exocytosis. However, protein kinase C (PKC) inhibitors decreased the facilitatory effects but not the inhibitory effects of AngII on stimulus-secretion coupling. The AngII type 1 receptor (AT1R) antagonist losartan blocked both inhibition and facilitation of secretion by AngII. The results of this study show that activation of multiple types of G-proteins and transduction pathways by a single neuromodulator acting through one receptor type can produce concentration-dependent, bi-directional regulation of exocytosis.

Adrenal Medulla↗

PubMed2000

P2Y purinoceptors inhibit exocytosis in adrenal chromaffin cells via modulation of voltage-operated calcium channels.

We have used combined membrane capacitance measurements (C(m)) and voltage-clamp recordings to examine the mechanisms underlying modulation of stimulus-secretion coupling by a G(i/o)-coupled purinoceptor (P2Y) in adrenal chromaffin cells. P2Y purinoceptors respond to extracellular ATP and are thought to provide an important inhibitory feedback regulation of catecholamine release from central and sympathetic neurons. Inhibition of neurosecretion by other G(i/o)-protein-coupled receptors may occur by either inhibition of voltage-operated Ca(2+) channels or modulation of the exocytotic machinery itself. In this study, we show that the P2Y purinoceptor agonist 2-methylthio ATP (2-MeSATP) significantly inhibits Ca(2+) entry and changes in C(m) evoked by single 200 msec depolarizations or a train of 20 msec depolarizations (2.5 Hz). We found that P2Y modulation of secretion declines during a train such that only approximately 50% of the modulatory effect remains at the end of a train. The inhibition of both Ca(2+) entry and DeltaC(m) are also attenuated by large depolarizing prepulses and treatment with pertussis toxin. Inhibition of N-type, and to lesser extent P/Q-type, Ca(2+) channels contribute to the modulation of exocytosis by 2-MeSATP. The Ca(2+)-dependence of exocytosis triggered by either single pulses or trains of depolarizations was unaffected by 2-MeSATP. When Ca(2+) channels were bypassed and exocytosis was evoked by flash photolysis of caged Ca(2+), the inhibitory effect of 2-MeSATP was not observed. Collectively, these data suggest that inhibition of exocytosis by G(i/o)-coupled P2Y purinoceptors results from inhibition of Ca(2+) channels and the Ca(2+) signal controlling exocytosis rather than a direct effect on the secretory machinery.

Adenosine Triphosphate↗

PubMed1999

Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon.

Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin-Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic beta cells, apparent resting values of cytosolic [Ca2+] and [Ca2+](gd) were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within approximately 1 microm of the plasma membrane underwent increases in [Ca2+](gd) that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes.

Animals↗

PubMed1999

Inhibition of the current of heterologously expressed HERG potassium channels by imipramine and amitriptyline.

1 Tricyclic antidepressants (TCAs) are associated with cardiovascular side effects including prolongation of the QT interval of the ECG. In this report we studied the effects of two TCAs (imipramine and amitriptyline) on ionic current mediated by cloned HERG potassium channels. 2 Voltage clamp measurements of HERG currents were made from CHO cells transiently transfected with HERG cDNA. HERG-encoded potassium channels were inhibited in a reversible manner by both imipramine and amitriptyline. HERG tail currents (IHERG) following test pulses to +20 mV were inhibited by imipramine with an IC50 of 3.4+/-0.4 microM (mean+/-s.e.mean) and a Hill coefficient of 1.17+/-0.03 (n = 5). 3 microM amitriptyline inhibited IHERG by 34+/-6% (n = 3). The inhibition showed only weak voltage dependence. 3 Using an 'envelope of tails' comprised of pulses to +20 mV of varying durations, the tau of activation was found to be 155+/-30 ms for control and 132+/-26 ms for 3 microM imipramine (n = 5). Once maximal channel activation was achieved after 320 ms (as demonstrated by maximal tail currents), further prolongation of depolarization did not increase imipramine-mediated HERG channel inhibition. 4 Taking current measurements every second during a 10 s depolarizing pulse from -80 mV to 0 mV, block was observed during the first pulse in the presence of imipramine and the level of IHERG block was similar throughout the pulse (n=5). 5 A three pulse protocol (two depolarizing pulses to +20 mV separated by 20 ms at -80 mV) revealed that imipramine did not significantly alter the kinetics of IHERG inactivation. The tau of inactivation was 8+/-2 ms and 5.6+/-0.4 ms (n = 5) in the absence and presence of 3 microM imipramine, respectively, and currents inactivated to a similar extent. 6 Our data are consistent with TCAs causing components of block of the HERG channel in both the closed and open states. Any component of open channel block occurs rapidly upon depolarization. Inhibition of IHERG by the prototype TCAs imipramine and amitriptyline may suggest a mechanism for QT prolongation associated with risks of arrhythmia and sudden death that accompany high concentrations of TCAs following overdose.

Amitriptyline↗

PubMed1998

Excitation-secretion coupling in mammalian neurohypophysial nerve terminals.

1. Oxytocin and vasopressin secretion from the neurohypophysis (NHP) is evoked by strongly patterned bursts of action potentials. We studied excitation-secretion coupling in single isolated terminals of rat NHP using patch clamp and capacitance detection techniques. 2. The secretory response evoked by trains of depolarizing pulses consisted of two discrete phases. Ca2+ entry during pulses early in the train did not elicit secretion. Exocytotic responses began only after a characteristic amount of total Ca2+ entry called "threshold". 3. In the postthreshold secretory phase, exocytotic events occurred during or immediately after depolarizing pulses, indicating that the final Ca(2+)-dependent step is triggered by high Ca2+ concentrations near the plasma membrane that dissipate rapidly after channel closure. Secretion was sensitive to both the concentration and species of Ca2+ chelator. BAPTA, a Ca2+ chelator with rapid Ca2+ binding kinetics, was more effective than EGTA in diminishing secretion. 4. The "threshold" amount of Ca2+ was determined by the concentration, but not species, of Ca2+ chelator. The threshold value was constant even when Ca2+ entry parameters were varied over a broad range of current amplitudes, pulse durations, and number of pulses, indicating that it did not require high Ca2+ concentrations near the plasma membrane. 5. These results suggest that the secretory response to a train of pulses consists of a Ca(2+)-dependent preparatory step that must be completed before subsequent Ca2+ entry can elicit exocytosis. 6. Exocytotic responses during single trains showed strong depression at a step subsequent to Ca2+ entry. Recovery from depression required 30-60 sec. 7. The properties of threshold secretion observed in NHP terminals are discussed in terms of current models of secretion.

Animals↗

PubMed1996

Domains of P2X receptors involved in desensitization.

ATP-gated ion channels (P2X receptors) are abundantly expressed in both neuronal and nonneuronal tissues, where they can serve as postsynaptic receptors. The response to ATP shows marked desensitization in some tissues but not others. Currents induced by ATP in Xenopus oocytes expressing cloned P2X1 (or P2X3) receptor had strong desensitization, whereas currents in cells expressing P2X2 receptors desensitized relatively little (90% vs. 14% decline of current in a 10-s application). In chimeric receptors, substitution into the P2X1 receptor of either one of two 34-residue segments from the P2X2 receptor removed the desensitization; these segments included the first or the second hydrophobic domain. In contrast, desensitization was introduced into the P2X2 receptor only by providing both these segments of the P2X1 (or P2X3) receptor. This suggests that desensitization requires interaction between the two hydrophobic domains of the receptor, and supports the view that these are membrane-spanning segments.

Adenosine Triphosphate↗

PubMed1996

Ba2+ ions evoke two kinetically distinct patterns of exocytosis in chromaffin cells, but not in neurohypophysial nerve terminals.

The coupling between divalent cations and exocytosis of large dense-cored vesicles (LDCV) was studied with capacitance-detection techniques in nerve terminals of the rat neurohypophysis (NHP) and bovine chromaffin cells. Ba2+ substitution for Ca2+ produced kinetically distinct responses in the two preparations. In NHP terminals, Ba2+ ions behave as weak substitutes for Ca2+. Exocytotic events occur principally during depolarizing pulses, i.e., events are "stimulus-coupled" to Ba2+ entry through voltage-gated Ca2+ channels. Stimulus-coupled exocytosis apparently requires elevated submembrane cation concentrations that dissipate rapidly on hyperpolarization-induced Ca(2+)-channel closure. Intracellular dialysis of NHP terminals with Ba2+ does not evoke exocytosis, nor does it interfere with depolarization-evoked Ca2+ influx and exocytosis. In chromaffin cells, Ba2+ ions evoke a small quantity of stimulus-coupled secretion, but the dominant response is an additional pronounced poststimulus capacitance increase that outlasts channel closures by 20-50 sec. "Stimulus-decoupled" exocytosis is slow (approximately 25-40 fF/sec) compared with Ca(2+)-evoked stimulus-coupled exocytosis (approximately 1000 fF/sec). Decoupled secretion is not attributable to Ba2+ displacement of intracellular Ca2+ ions, because it is insensitive to 10 mM EGTA or thapsigargin. Slow exocytosis is initiated by inclusion of Ba2+ ions in the recording pipette and continues steadily for 5-12 min, producing a total increase of several thousand fF, which ultimately doubles or triples the original cell-surface area. We propose that two pathways of regulated exocytosis with distinct kinetics and divalent cation sensitivity exist in chromaffin cells. Only a single kinetic pattern is detected in NHP terminals, suggesting that mechanisms for secretion are not universally distributed in excitable cells.

Animals↗

PubMed1996

Kinetics of stimulus-coupled secretion in dialyzed bovine chromaffin cells in response to trains of depolarizing pulses.

Stimulus-secretion coupling in bovine chromaffin cells was investigated with whole-cell patch-clamp recordings and capacitance detection techniques to monitor exocytosis in response to trains of depolarizing pulses. Two kinetically discrete modes of exocytotic responses were observed. In one mode, the first depolarization of a train elicited a large increase in membrane capacitance (Cm; mean approximately 70 fF). This secretory mode was characterized by small Ca2+ requirements, relative insensitivity to the pipette Ca2+ chelator concentration, and rapid depletion of the secretory response. This mode of stimulus-secretion coupling was labile and was seen only in response to the first and, occasionally, the second stimulus train of whole-cell recordings. The second type of exocytotic response persisted for the remainder of the whole-cell recordings and consisted of two distinct phases. During the earliest pulses of a stimulus train, Ca2+ entry did not evoke Cm increases. Instead, Cm responses were elicited by later pulses, despite diminished Ca2+ entry per pulse caused by Ca2+ channel inactivation. The secretory phase was initiated after a specific "threshold" amount of Ca2+ had entered the cell, which was determined by the concentration, but not the binding kinetics, of the Ca2+ chelator in the pipette. In both the early and the secretory phases, the response of the cell was proportional to cumulative Ca2+ entry, regardless of current amplitude, pulse duration, or number of pulses. Threshold-type secretory kinetics has been described previously in peptide-secreting neurohypophysial (NHP) nerve terminals (Seward et al., 1995). Secretory kinetics with minimal Ca2+ requirements has not been observed in that preparation. Chromaffin cells appear to possess a broader repertoire of stimulus-secretion coupling modes than NHP terminals.

Action Potentials↗

PubMed1996

Coexpression with potassium channel subunits used to clone the Y2 receptor for neuropeptide Y.

Xenopus oocytes were injected with RNAs for the two inward-rectifier potassium channel subunits Kir3.1 (GIRK1) and Kir3.4 (rcKATP or CIR) in addition to RNA from the neuroblastoma cell line KAN-TS. Potassium currents were evoked by neuropeptide Y in oocytes injected with polyadenylated RNA or with cRNA from pools of a neuroblastoma (KAN-TS) cDNA library, and progressive subdivision of responding pools yielded a single cDNA. The encoded protein contains 381 amino acids, has the seven hydrophobic domains characteristic of G protein-coupled receptors, and is 31% identical to the Y1 receptor: potassium currents were induced by neuropeptide Y (EC50=60pm) and Y2-selective analogues. Coexpression with potassium channel subunits will be a generally useful method for the cloning of G protein-coupled receptors.

Amino Acid Sequence↗

PubMed1996

Functional selectivity of orphanin FQ for its receptor coexpressed with potassium channel subunits in Xenopus laevis oocytes.

An opioid-like receptor has been cloned by several groups of researchers and recently shown to be activated by an endogenous heptadecapeptide termed orphanin FQ (or nociceptin). We isolated the corresponding mouse cDNA and coexpressed it in Xenopus laevis oocytes with the potassium channel subunits Kir3.1 (GIRK1) and Kir3.4 (CIR, rcKATP). Orphanin FQ evoked potassium currents, with 50% of the maximal effect at approximately 1 nM; [Tyr1]orphanin FQ was equally effective, and des-pheorphanin FQ was without activity. Dynorphin A, dynorphin(1-9), dynorphin(1-13), and alpha-neoendorphin were > 100 times less potent, and other agonists active at mu-, delta-, and kappa-opioid receptors had no effect. Naloxone (1 microM) and norbinaltorphimine (1 microM) had no antagonist action. Conversely, oocytes expressing kappa receptors responded to dynorphin (half-maximal concentration, 0.3 nM) but not to orphanin FQ. Thus, both kappa and orphanin FQ receptors readily couple to potassium channels, but the highly selective activation by dynorphin and orphanin FQ is consistent with distinct functional pathways in vivo.

Amino Acid Sequence↗

PubMed1995

Activation of nicotinic receptors triggers exocytosis from bovine chromaffin cells in the absence of membrane depolarization.

The traditional function of neurotransmitter-gated ion channels is to induce rapid changes in electrical activity. Channels that are Ca(2+)-permeable, such as N-methyl-D-aspartate receptors at depolarized membrane potentials, can have a broader repertoire of consequences, including changes in synaptic efficacy, developmental plasticity, and excitotoxicity. Neuronal nicotinic receptors for acetylcholine (nAChRs) are usually less Ca(2+)-permeable than N-methyl-D-aspartate receptors but have a significant Ca2+ permeability, which is greater at negative potentials. Here we report that in neuroendocrine cells, activation of nAChRs can trigger exocytosis at hyperpolarized potentials. We used whole-cell patch-clamp recordings to record currents and the capacitance detection technique to monitor exocytosis in isolated bovine chromaffin cells. Stimulation of nAChRs at hyperpolarized potentials (-60 or -90 mV) evokes a large current and a maximal capacitance increase corresponding to the fusion of approximately 200 large dense-core vesicles. The amount of exocytosis is controlled both by the Ca2+ influx through nAChRs and by a contribution from thapsigargin-sensitive Ca2+ sequestering stores. This is a form of neurotransmitter action in which activation of nAChRs triggers secretion through an additional coupling pathway that coexists with classical voltage-dependent Ca2+ entry.

Adrenal Medulla↗

PubMed1995

Chromaffin cell cortical actin network dynamics control the size of the release-ready vesicle pool and the initial rate of exocytosis.

Morphological, biochemical, and membrane capacitance measurements were used to study the role of cortical filamentous actin (F-actin) in exocytosis. Fluorescence and electron microscopy of resting chromaffin cells revealed a cortical actin network that excluded secretory vesicles from the subplasmalemmal area. Phorbol ester (PMA) treatment disrupted cortical F-actin and increased both the number of vesicles within the 0-50 nm subplasmalemmal zone and the initial rate of stimulated catecholamine release. In PMA-pretreated cells, membrane capacitance studies showed an increased number of vesicles fusing with the plasmalemma during the first two depolarizations of a train. PMA did not affect voltage-dependent Ca2+ influx. The total number of vesicles fused with the plasma membrane correlated well with the number of vesicles occupying the 0-50 nm cortical zone. Therefore, cortical F-actin disassembly allows translocation of vesicles to the plasmalemma in preparation for exocytosis.

Actins↗

PubMed1995

Exocytosis in peptidergic nerve terminals exhibits two calcium-sensitive phases during pulsatile calcium entry.

The link between electrical activity, Ca2+ entry through voltage-gated channels, and transmitter or hormone secretion is a central issue in neurobiology. In peptidergic nerve terminals of the mammalian neurohypophysis (NHP), secretion is elicited by patterned bursts of action potentials (APs). All parameters of the bursts are important to elicit efficient secretion, including AP frequency, AP broadening, burst duration, and interburst interval (Leng, 1988). We have studied Ca(2+)-secretion coupling of peptide-containing large dense-core vesicles (LDCV) in isolated rat NHP terminals. Ca2+ influx through voltage-gated Ca2+ channels was elicited and recorded by the whole-cell patch-clamp technique. Exocytosis was monitored on line with high temporal resolution by the capacitance detection technique (Neher and Marty, 1982). AP bursts were simulated by depolarizing pulse trains that mimic pulsatile submembrane Ca2+ elevations predicted for physiological stimuli. The characteristic capacitance response (delta Cm) to a train of depolarizing pulses was triphasic. It consisted of a threshold phase during which early pulses did not elicit secretion, a subsequent secretory phase during which Cm increases were coupled to depolarizing pulses, and a fatigued or inactivated state during which additional Ca2+ entry was ineffective. Both the threshold phase and secretory phase were correlated with the integrals of Ca2+ current. Ca2+ chelators affect both the threshold and secretory phase at submillimolar concentrations. Thus, a "shell" rather than "microdomain" model of Ca2+ elevation is appropriate for analyzing Ca(2+)-secretion coupling in NHP terminals (Nowycky and Pinter, 1993). We propose a two-step model, with a ca(2+)-dependent preparatory step followed by a final exocytotic step that is coupled to active Ca2+ influx. The results suggest that under physiological conditions, APs early in a burst prepare an NHP terminal for secretion, but later APs actually trigger exocytosis. Since NHP terminals do not possess a readily releasable pool of vesicles that require only a single Ca2+ step for exocytosis as seen in chromaffin cells (Neher and Zucker, 1993) and melanotrophs (Thomas et al, 1993a), Ca(2+)-secretion coupling mechanisms may be heterologous even within a single class of vesicles.

Action Potentials↗

PubMed1990

Characterization of two components of the N-like, high-threshold-activated calcium channel current in differentiated SH-SY5Y cells.

Retinoic acid differentiated SH-SY5Y cells exhibit only a high-threshold-activated (-30 to -20 mV) whole cell calcium channel current. When barium was used as the charge carrier, the high-threshold-activated current showed bi-exponential inactivation kinetics during a 500 ms voltage step from -90 to +10 mV. The time constants of inactivation were approximately 75 and 750 ms. The fast inactivating component was more sensitive than the slow inactivating component to steady-state inactivation at depolarized holding potentials. The calcium channel current was inhibited by externally applied cadmium (10-300 microM) and gadolinium (10-30 microM) as well as by high concentrations of nickel and cobalt. omega Conus toxin (1 microM) irreversibly blocked the calcium channel current. However, the dihydropyridine agonist, BAY K 8644 (3-10 microM) and antagonists, nifedipine (3-10 microM) and nimodipine (10 microM) did not affect either component of the calcium channel current. Agents which blocked the calcium channel current did not exhibit any selectivity for the fast inactivating over the slow inactivating component of the current. These results indicate that whilst the calcium channel current recorded in differentiated SH-SY5Y cells can be classified on the basis of the blocking agents as being of the N type, the current shows more than one form of inactivation.

Calcium Channel Agonists↗