Electrophysiological Investigation of the Subcellular Fine Tuning of Sympathetic Neurons by Hydrogen Sulfide.

H2S is well-known as hypotensive agent, whether it is synthetized endogenously or administered systemically. Moreover, the H2S donor NaHS has been shown to inhibit vasopressor responses triggered by stimulation of preganglionic sympathetic fibers. In contradiction with this latter result, NaHS has been reported to facilitate transmission within sympathetic ganglia. To resolve this inconsistency, H2S and NaHS were applied to primary cultures of dissociated sympathetic ganglia to reveal how this gasotransmitter might act at different subcellular compartments of such neurons. At the somatodendritic region of ganglionic neurons, NaHS raised the frequency, but not the amplitudes, of cholinergic miniature postsynaptic currents via a presynaptic site of action. In addition, the H2S donor as well as H2S itself caused membrane hyperpolarization and decreased action potential firing in response to current injection. Submillimolar NaHS concentrations did not affect currents through Kυ7 channels, but did evoke currents through K ATP channels. Similarly to NaHS, the K ATP channel activator diazoxide led to hyperpolarization and decreased membrane excitability; the effects of both, NaHS and diazoxide, were prevented by the K ATP channel blocker tolbutamide. At postganglionic sympathetic nerve terminals, H2S and NaHS enhanced noradrenaline release due to a direct action at the level of vesicle exocytosis. Taken together, H2S may facilitate transmitter release within sympathetic ganglia and at sympatho-effector junctions, but causes hyperpolarization and reduced membrane excitability in ganglionic neurons. As this latter action was due to K ATP channel gating, this channel family is hereby established as another previously unrecognized determinant in the function of sympathetic ganglia.

Excitation of rat sympathetic neurons via M1 muscarinic receptors independently of Kv7 channels.

The slow cholinergic transmission in autonomic ganglia is known to be mediated by an inhibition of Kv7 channels via M1 muscarinic acetylcholine receptors. However, in the present experiments using primary cultures of rat superior cervical ganglion neurons, the extent of depolarisation caused by the M1 receptor agonist oxotremorine M did not correlate with the extent of Kv7 channel inhibition in the very same neuron. This observation triggered a search for additional mechanisms. As the activation of M1 receptors leads to a boost in protein kinase C (PKC) activity in sympathetic neurons, various PKC enzymes were inhibited by different means. Interference with classical PKC isoforms led to reductions in depolarisations and in noradrenaline release elicited by oxotremorine M, but left the Kv7 channel inhibition by the muscarinic agonist unchanged. M1 receptor-induced depolarisations were also altered when extra- or intracellular Cl(-) concentrations were changed, as were depolarising responses to γ-aminobutyric acid. Depolarisations and noradrenaline release triggered by oxotremorine M were reduced by the non-selective Cl(-) channel blockers 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid and niflumic acid. Oxotremorine M induced slowly rising inward currents at negative membrane potentials that were blocked by inhibitors of Ca(2+)-activated Cl(-) and TMEM16A channels and attenuated by PKC inhibitors. These channel blockers also reduced oxotremorine M-evoked noradrenaline release. Together, these results reveal that slow cholinergic excitation of sympathetic neurons involves the activation of classical PKCs and of Ca(2+)-activated Cl(-) channels in addition to the well-known inhibition of Kv7 channels.

Constitutive activity of the A2A adenosine receptor and compartmentalised cyclic AMP signalling fine-tune noradrenaline release.

Neuroblastoma SH-SY5Y (SH) cells endogenously express A(2A) adenosine receptors and can be differentiated into a sympathetic neuronal phenotype, capable of depolarisation-dependent noradrenaline release. Using differentiated SH culture, we here explored the link between A(2A)-receptor signalling and neurotransmitter release. In response to the receptor agonist CGS21680, the cells produced cyclic AMP (cAMP), and when depolarised, they released increased amounts of noradrenaline. An A(2A)-receptor antagonist, XAC, as well as an inhibitor of cAMP-dependent protein kinase A (PKA), H89, depressed agonist-dependent release. In the presence of XAC or H89, noradrenaline release was found to be below basal values. This suggested that release facilitation also owes to constitutive receptor activity. We demonstrate that even in the absence of an agonist, the native A(2A)-receptor stimulated cAMP production, leading to the activation of PKA and enhanced noradrenaline release. Ancillary, non-cAMP-dependent effects of the receptor (i.e. phosphorylation of CREB, of Rabphilin3A) were refractory to constitutive activation. PKA-dependent facilitation of noradrenaline release was recapitulated with membrane-permeable 8-Br-cAMP; in addition to facilitation, 8-Br-cAMP caused marked inhibition of release, an effect not observed upon receptor activation. Inhibition by receptor-independent cAMP was likely due to suppression of voltage-dependent calcium current (VDCC) and increased activity of Src-family kinases. Receptor-mediated release facilitation was reproduced in the presence of tetrodotoxin (blocking action potentials); hence, the signalling occurred at the active zone comprising release sites. Our findings thus support (1) presynaptic localisation of the A(2A)-receptor and (2) suggest that compartmentalised pathways transmit cAMP signalling in order to facilitate depolarisation-dependent neurotransmitter release.

Facilitation of transmitter release from rat sympathetic neurons via presynaptic P2Y(1) receptors.

BACKGROUND AND PURPOSE: P2Y(1) , P2Y(2) , P2Y(4) , P2Y(12) and P2Y(13) receptors for nucleotides have been reported to mediate presynaptic inhibition, but unequivocal evidence for facilitatory presynaptic P2Y receptors is not available. The search for such receptors was the purpose of this study. EXPERIMENTAL APPROACH: In primary cultures of rat superior cervical ganglion neurons and in PC12 cell cultures, currents were recorded via the perforated patch clamp technique, and the release of [(3) H]-noradrenaline was determined. KEY RESULTS: ADP, 2-methylthio-ATP and ATP enhanced stimulation-evoked (3) H overflow from superior cervical ganglion neurons, treated with pertussis toxin to prevent the signalling of inhibitory G proteins. This effect was abolished by P2Y(1) antagonists and by inhibition of phospholipase C, but not by inhibition of protein kinase C or depletion of intracellular Ca(2+) stores. ADP and a specific P2Y(1) agonist caused inhibition of Kv7 channels, and this was prevented by a respective antagonist. In neurons not treated with pertussis toxin, (3) H overflow was also enhanced by a specific P2Y(1) agonist and by ADP, but only when the P2Y(12) receptors were blocked. ADP also enhanced K(+) -evoked (3) H overflow from PC12 cells treated with pertussis toxin, but only in a clone expressing recombinant P2Y(1) receptors. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that presynaptic P2Y(1) receptors mediate facilitation of transmitter release from sympathetic neurons most likely through inhibition of Kv7 channels.

Differential fading of inhibitory and excitatory B2 bradykinin receptor responses in rat sympathetic neurons: a role for protein kinase C.

Through inhibitory and excitatory effects on sympathetic neurons, B(2) bradykinin receptors contribute to protective and noxious cardiovascular mechanisms. Presynaptic inhibition of sympathetic transmitter release involves an inhibition of Ca(V)2 channels, neuronal excitation an inhibition of K(V)7 channels. To investigate which of these mechanisms prevail over time, the respective currents were determined. The inhibition of Ca(2+) currents by bradykinin reached a maximum of 50%, started to fade within the first minute, and became attenuated significantly after > or = 4 min. The inhibition of K(+) currents reached a maximum of 85%, started to fade after > 3 min, and became attenuated significantly after > or = 7 min. Blocking Ca(2+)-independent protein kinase C (PKC) enhanced the inhibition of Ca(2+) currents by bradykinin and delayed its fading, left the inhibition of K(+) currents and its fading unaltered, and enhanced the reduction of noradrenaline release and slowed its fading. Conversely, direct activation of PKC abolished the inhibition of noradrenaline release and largely attenuated the inhibition of Ca(2+) currents. These results show that the inhibitory effects of bradykinin in sympathetic neurons are outweighed over time by its excitatory actions because of more rapid, PKC-dependent fading of the inhibitory response.

Presynaptic inhibition via a phospholipase C- and phosphatidylinositol bisphosphate-dependent regulation of neuronal Ca2+ channels.

Presynaptic inhibition of transmitter release is commonly mediated by a direct interaction between G protein betagamma subunits and voltage-activated Ca2+ channels. To search for an alternative pathway, the mechanisms by which presynaptic bradykinin receptors mediate an inhibition of noradrenaline release from rat superior cervical ganglion neurons were investigated. The peptide reduced noradrenaline release triggered by K+-depolarization but not that evoked by ATP, with Ca2+ channels being blocked by Cd2+. Bradykinin also reduced Ca2+ current amplitudes measured at neuronal somata, and this effect was pertussis toxin-insensitive, voltage-independent, and developed slowly within 1 min. The inhibition of Ca2+ currents was abolished by a phospholipase C inhibitor, but it was not altered by a phospholipase A2 inhibitor, by the depletion of intracellular Ca2+ stores, or by the inactivation of protein kinase C or Rho proteins. In whole-cell recordings, the reduction of Ca2+ currents was irreversible but became reversible when 4 mM ATP or 0.2 mM dioctanoyl phosphatidylinositol-4,5-bisphosphate was included in the pipette solution. In contrast, the effect of bradykinin was entirely reversible in perforated-patch recordings but became irreversible when the resynthesis of phosphatidylinositol-4,5-bisphosphate was blocked. Thus, the inhibition of Ca2+ currents by bradykinin involved a consumption of phosphatidylinositol-4,5-bisphosphate by phospholipase C but no downstream effectors of this enzyme. The reduction of noradrenaline release by bradykinin was also abolished by the inhibition of phospholipase C or of the resynthesis of phosphatidylinositol-4,5-bisphosphate. These results show that the presynaptic inhibition was mediated by a closure of voltage-gated Ca2+ channels through depletion of membrane phosphatidylinositol bisphosphates via phospholipase C.

Antibodies directed against Lewis-Y antigen inhibit signaling of Lewis-Y modified ErbB receptors.

The majority of cancer cells derived from epithelial tissue express Lewis-Y (LeY) type difucosylated oligosaccharides on their plasma membrane. This results in the modification of cell surface receptors by the LeY antigen. We used the epidermal growth factor (EGF) receptor family members ErbB1 and ErbB2 as model systems to investigate whether the sugar moiety can be exploited to block signaling by growth factor receptors in human tumor cells (i.e., SKBR-3 and A431, derived from a breast cancer and a vulval carcinoma, respectively). The monoclonal anti-LeY antibody ABL364 and its humanized version IGN311 immunoprecipitated ErbB1 and ErbB2 from detergent lysates of A431 and SKBR-3, respectively. ABL364 and IGN311 blocked EGF- and heregulin-stimulated phosphorylation of mitogen-activated protein kinase [MAPK = extracellular signal-regulated kinase 1/2] in SKBR-3 and A431 cells. The effect was comparable in magnitude with that of trastuzumab (Herceptin) and apparently noncompetitive with respect to EGF. Stimulation of MAPK by ErbB was dynamin dependent and contingent on receptor internalization. ABL364 and IGN311 changed the intracellular localization of fluorescent EGF-containing endosomes and accelerated recycling of intracellular [(125)I]EGF to the plasma membrane. Taken together, these observations show that antibodies directed against carbohydrate side chains of ErbB receptors are capable of inhibiting ErbB-mediated signaling. The ability of these antibodies to reroute receptor trafficking provides a mechanistic explanation for their inhibitory action.

Nicotinic acid-adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor.

Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca(2+)-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca(2+)-release from intracellular Ca(2+) stores can be triggered by diffusible second messengers like Ins P (3), cyclic ADP-ribose or nicotinic acid-adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca(2+)-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca(2+)-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca(2+)-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC(50) approximately 30 nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25 nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel.