Peter F. T. Vaughan

The regulation of neurotransmitter secretion by protein kinase C

The effect of protein kinase C(PKC) on the release of neurotransmitters from a number preparations, including sympathetic nerve endings, brain slices, synaptosomes, and neuronally derived cell lines, is considered. A comparison is drawn between effects of activation of PKC on neurotransmitter release from small synaptic vesicles and large dense-cored vesicles. The enhancement of neurotransmitter release is discussed in relation to the effect of PKC on:1.Rearrangement of the F-actin-based cytoskeleton, including the possible role of MARCKS in this process, to allow access of large dense-cored vesicles to release sites on the plasma membrane.2.Phosphorylation of key components in the SNAP/SNARE complex associated with the docking and fusion of vesicles at site of secretion.3.Ion channel activity, particularly Ca2+ channels.

Differential effects of unaggregated and aggregated amyloid β protein (1–40) on K+ channel currents in primary cultures of rat cerebellar granule and cortical neurones

The effects of amyloid β protein on voltage‐gated K+ channel currents were studied using the whole‐cell patch‐clamp technique. The 1–40 amino acid form of amyloid β protein was applied to primary cultures of rat cerebellar granule and cortical neurones for 24 h. Both the unaggregated and aggregated forms of the peptide, which have differing biological activities, were used. In cerebellar granule neurones, 24‐h pre‐incubation with 1 µm unaggregated amyloid β protein resulted in a 60% increase in the ‘A’‐type component of K+ current. Increased delayed rectifier activity was Cd2+‐sensitive and was presumed to be secondary to an increase in voltage‐gated Ca2+ channel current activity. Unaggregated amyloid β protein had no effect on any component of the K+ channel current in cortical neurones. One micromolar of aggregated amyloid β protein had no effect on K+ channel current in either cell type but reduced cell survival within 24 h as measured using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assays. The unaggregated form of amyloid β protein had no neurotoxic effects when applied to either neurone type for up to 72 h. These data indicate that the unaggregated, non‐pathological form of amyloid β protein causes changes in the ion channel function of neurones, possibly reflecting a physiological role for the peptide.

The use of the human neuroblastoma SH-SY5Y to study the effect of second messengers on noradrenaline release

1. Recent data suggesting that the human neuroblastoma SH-SY5Y is a suitable cell line in which to study the effect of second messengers on NA release are discussed in the context of current views on exocytosis. 2. Release of NA is evoked by depolarization, as well as activation of muscarinic (M3) and bradykinin (B2) receptors in SH-SY5Y cells which have not been differentiated by the addition of growth factors. 3. Evoked release is enhanced by activation of protein kinase C. 4. Activation of protein kinase C decreases the changes in intracellular calcium evoked by carbachol, bradykinin and 100 mM K+. 5. SH-SY5Y express N-type and L-type voltage sensitive Ca2+ channels. L-Type Ca(2+)-channels are coupled to NA release under conditions of weak depolarization. However with strong depolarization (100 mM K+) both L-type and N-type channels are involved. 6. Muscarinic- and neuropeptide Y receptors are coupled to the inhibition of Ca2+ channel activity.

Electrical stimulation of the vagus increases extracellular glutamate recovered from the nucleus tractus solitarii of the cat by in vivo microdialysis

Release of glutamate into the extracellular space of the cat nucleus tractus solitarii (NTS) was measured by in vivo microdialysis and high performance liquid chromatography. Perfusion of the probe with 100 mM potassium increased glutamate release by 211% (P < 0.001), while electrical stimulation of the cervical vagus increased release by 53% (P < 0.01). These results are compatible with the hypothesis that glutamate is a neurotransmitter released by vagal afferent nerve terminals in the NTS.

Inhibition of neuronal nicotinic acetylcholine receptors by imipramine and desipramine

The actions of two structurally related tricyclic antidepressants on neuronal nicotinic acetylcholine receptors were investigated in human neuroblastoma (SY-SY5Y) cells, using whole-cell patch-clamp recordings. Both desipramine and imipramine reversibly inhibited inward currents evoked by application of the nicotinic receptor agonist dimethylphenylpiperazinium iodide (30-300 microM) with IC50 values of 0.17 microM and 1.0 microM respectively (holding potential -70 mV). The degree of current inhibition caused by either tricyclic compound was unaffected by agonist concentration (30-300 microM). The effects of desipramine were voltage-independent over the range -40 mV to -100 mV, and inhibition caused by imipramine only increased very slightly with membrane hyperpolarization over the same range. These results indicate that tricyclic antidepressants can inhibit neuronal nicotinic acetylcholine receptors by mechanisms which are distinct from their actions at non-neuronal nicotinic acetylcholine receptors.

Glibenclamide inhibits a voltage-gated K+ current in the human neuroblastoma cell line SH-SY5Y

The whole-cell patch-clamp technique was used to record outward K+ currents in the human neuroblastoma cell line SH-SY5Y. These K+ currents were inhibited by tetraethylammonium (20 mM) and by 4-aminopyridine (2 mM). The Ca2+ channel blocker Cd2+ (0.2 mM) also inhibited K+ currents, indicating that a component was Ca(2+)-activated. Glibenclamide, a presumed selective inhibitor of ATP-sensitive K+ channels, caused reversible inhibitions of the K+ currents and appeared to accelerate their inactivation. These effects were not significantly affected by intracellular ATP (ATPi), and were observed in the presence of 0.2 mM Cd2+. It is concluded that glibenclamide inhibits a voltage-gated, Ca(2+)- and ATPi-independent K+ current in SH-SY5Y cells.

Potassium- and Carbachol-Evoked Release of [3H]Noradrenaline from Human Neuroblastoma Cells, SH-SY5Y

Abstract: The human neuroblastoma clone SH‐SY5Y expresses potassium‐, carbachol‐, and calcium ionophore A23187‐evoked, calcium‐dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki= 0.33 nM), hexahydrosiladifenidol (Ki= 18 nM), and pirenzepine (Ki= 1,183 nM) completely inhibited the carbachol‐evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2‐selective antagonists meth‐octramine (10‐4M) and AFDX‐116 (10‐4M), by ∼14 and 46%, respectively. The nicotinic antagonist d‐tubocurarine (10‐4M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH‐SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.

Calcium Channel Currents in Undifferentiated Human Neuroblastoma (SH‐SY5Y) Cells: Actions and Possible Interactions of Dihydropyridines and ω‐Conotoxin

Ca2+ channel currents were recorded in undifferentiated human neuroblastoma (SH‐SY5Y) cells with the whole‐cell patch‐clamp technique, using 10 mM Ba2+ as charge carrier. Currents were only evoked by depolarizations to ‐30 mV or more positive (holding potential ‐80 mV), inactivated partially during 200 ms depolarizing steps, and were abolished by 150 μMCd2+. Currents could be enhanced by Bay K‐8644 and partially inhibited by nifedipine, suggesting that they arose in part due to activation of L‐type Ca2+ channels. Currents were also inhibited by the marine snail peptide ω‐conotoxin GVIA (ω‐CgTx). At a concentration of 10 nM inhibition by ω‐CgTx was reversible, but at higher concentrations blockade was always irreversible. Although current inhibition by nifedipine was maximal at 1μM, supramaximal concentrations reduced the inhibitory actions of ω‐CgTx in a concentration‐dependent manner. Ca2+ channel currents evoked from a holding potential of ‐50 mV showed no inactivation during 200 ms depolarizations but declined in amplitude with successive depolarizing steps (0.2 Hz). Current amplitudes could be restored by returning the holding potential to ‐80 mV. Currents evoked from ‐50 mV were inhibited by nifedipine and ω‐CgTx to a similar degree as those evoked from ‐80 mV. Our results indicate that undifferentiated SH‐SY5Y cells possess L‐ and N‐type Ca2+ channels which can be distinguished pharmacologically but cannot be separated by using depolarized holding potentials. Furthermore, these data suggest that nifedipine has a novel action to inhibit blockade of N‐type channels by ω‐CgTx.

Occurrence of two types of secretory vesicles in the human neuroblastoma SH-SY5Y.

Abstract: Western blot analysis showed that the human neuroblastoma SH‐SY5Y expresses the proteins synaptotagmin I, synaptobrevin, synapsin I, rab3a, syntaxin, SNAP‐25, NSF, α‐SNAP, and munc‐18, which have been implicated in the movement, docking, and fusion of vesicles during exocytosis from other neuroendocrine cells. The subcellular localization of secretogranins I and II, synaptotagmin I, neuropeptide Y, rab3a, synaptobrevin, synaptophysin, and syntaxin was investigated by immunofluorescence microscopy and revealed punctate staining patterns characteristic of secretory vesicles. The comigration of noradrenaline, secretogranin II, and dopamine‐β‐hydroxylase on sucrose‐D2O gradient fractions indicates the presence of a population of noradrenaline‐containing large dense‐cored vesicles (LDCVs). In addition, a lighter vesicle population is also present that does not appear to be noradrenergic and contains a 48‐kDa synaptophysin antigen absent from the large dense‐cored vesicles. Immunocytochemical experiments show that not all of the vesicles that express synaptotagmin I contain secretogranin II. Thus, our studies suggest that two types of vesicle are present in SH‐SY5Y cells, one of which, the LDCVs, contains noradrenaline. These findings confirm our previous studies suggesting that depolarization‐evoked release of noradrenaline from SH‐SY5Y occurs by LDCV exocytosis. This enhances the value of SH‐SY5Y as a cell line in which to study the mechanism by which noradrenaline release is regulated.

Activation of Protein Kinase C‐α and Translocation of the Myristoylated Alanine‐Rich C‐Kinase Substrate Correlate with Phorbol Ester‐Enhanced Noradrenaline Release from SH‐SY5Y Human Neuroblastoma Cells

Abstract: The aim of this study was to investigate the mechanism by which short‐term pretreatment with the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA; 100 nM) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH‐SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8‐min TPA treatment caused translocation of the α‐subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC‐ε from cytosolic and membrane‐associated to cytoskeleton‐ and membrane‐associated TPA had no effect on the cytosolic location of PKC‐ζ. Subcellular fractionation studies also showed that the myristoylated alanine‐rich C‐kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8‐min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31‐8220 (10 µM). Selective down‐regulation of PKC subtypes by prolonged exposure to phorbol 12,13‐dibutyrate (100 nM) attenuated the TPA‐induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down‐regulation of PKC‐α (but not ‐ε or ‐ζ). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH‐SY5Y cells due to the TPA‐induced activation of PKC‐α.