Presynaptic Inhibition via a Phospholipase C- and Phosphatidylinositol Bisphosphate-Dependent Regulation of Neuronal Ca Channels

Presynaptic inhibition of transmitter release is commonly mediated by a direct interaction between G protein subunits and voltage-activated Ca 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 Ca channels being blocked by Cd . Bradykinin also reduced Ca current amplitudes measured at neuronal somata, and this effect was pertussis toxin-insensitive, voltage-independent, and developed slowly within 1 min. The inhibition of Ca currents was abolished by a phospholipase C inhibitor, but it was not altered by a phospholipase A2 inhibitor, by the depletion of intracellular Ca stores, or by the inactivation of protein kinase C or Rho proteins. In whole-cell recordings, the reduction of Ca 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 Ca 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 Ca channels through depletion of membrane phosphatidylinositol bisphosphates via phospholipase C. Via changes in the strength of synaptic transmission, the nervous system can adapt to alterations in the environment, a phenomenon that is generally referred to as neuromodulation. In this respect, the modulation of transmitter release via presynaptic receptors is of utmost importance, and a plethora of neuromodulators act via presynaptic G proteincoupled receptors (GPCR). In most, if not all, types of synapses, the activation of GPCRs was found to lead to a presynaptic inhibition of transmitter release, because activated G protein subunits directly interacted with voltage-activated Ca channels (VACCs) and thereby reduced the Ca influx required for vesicle exocytosis (Stevens, 2004). The precise mechanisms underlying the modulation of VACCs via GPCRs have been investigated in greatest detail in sympathetic neurons (Hille, 1994); there, the receptor-dependent activation of G proteins leads to an inhibition of Ca currents (ICa) either via a direct, membrane-delimited and voltage-dependent interaction of G protein subunits with VACCs or via a second messenger system (Hille, 1994; Ikeda and Dunlap, 1999; Elmslie, 2003). In the experiments presented below, we used sympathetic neurons to delineate an This study was supported by the “Virologiefonds” of the Medical University of Vienna and by the Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung P15797 and P17611). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.014886. ABBREVIATIONS: GPCR, G protein-coupled receptor; ICa, Ca 2 current; DEDA, 7,7-dimethyl-5,8-eicosadienoic acid; diC8-PIP2, dioctanoyl phosphatidyl-4,5-bisphosphate; GF 109203X, bisindolylmaleimide I; IP3, inositol trisphosphate; KM channel, M-type K channel; LY 294,002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PIP2, phosphatidylinositol 4,5-bisphosphate; PI3-kinase, phosphatidylinositol 3-kinase; PI4kinase, phosphatidylinositol 4-kinase; PKC, protein kinase C; DTT, dithiothreitol; PAO, phenylarsine oxide; DMSO, dimethyl sulfoxide; PLA2, phospholipase A2; PLC, phospholipase C; PMA, phorbol-12-myristate-13-acetate; PTX, pertussis toxin; SCG, superior cervical ganglion; TTX, tetrodotoxin; U73122, 1-[6-[[(17 )-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; U73343, 1-[6-[[(17 )-3-methoxyestra1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrollidinedione; VACC, voltage-activated Ca channel; UK 14304, 5-bromo-N-(4,5-dihydro-1H-imidazol2-yl)-6-quinoxalinamine; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester. 0026-895X/05/6805-1387–1396$20.00 MOLECULAR PHARMACOLOGY Vol. 68, No. 5 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 14886/3059418 Mol Pharmacol 68:1387–1396, 2005 Printed in U.S.A. 1387 at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from example of presynaptic inhibition that relies on the modulation of VACCs through a second messenger system, but independently of a membrane-delimited action of G protein subunits. In rat superior cervical ganglion (SCG) neurons, a large number of GPCRs including the prototypic 2-adrenoceptors mediate the voltage-dependent, membrane-delimited, mediated inhibition of ICa, but only M1 muscarinic and AT1 angiotensin receptors were reported to reduce ICa in a voltage-independent manner via diffusible second messengers (Hille, 1994; Ikeda and Dunlap, 1999; Elmslie et al., 2003). These two latter receptors also use second messengers to inhibit M-type K (KM) channels (Hille, 1994). The underlying signal cascade remained obscure for decades but was recently evidenced to involve a phospholipase C (PLC)-dependent regulation of the membrane levels of phosphatidylinositol 4,5-bisphosphates (PIP2; Suh and Hille, 2002; Zhang et al., 2003; Winks et al., 2005). While the present work was in progress, the same signaling pathway was reported to mediate the inhibition of VACCs in SCG neurons via M1 receptors (Gamper et al., 2004). An inhibition of KM channels in SCG neurons has also been observed when B2 bradykinin receptors were activated (Jones et al., 1995), and this effect involved both the reduction of membrane PIP2 and inositol trisphophate-dependent increases in intracellular Ca (Cruzblanca et al., 1998; Bofill-Cardona et al., 2000; Winks et al., 2005). Most recently, we found that bradykinin also caused a reduction of transmitter release from SCG neurons via presynaptic B2 receptors and an inhibition of ICa (Edelbauer et al., 2005). However, the signaling cascade mediating the modulation of VACCs was not elucidated, and it remained obscure whether the inhibition of VACCs also occurred at presynaptic sites and thus was the basis for the reduction of transmitter release. Here, we first demonstrate that the inhibition of presynaptic VACCs is involved in the repression of transmitter release from rat SCG neurons by bradykinin and then provide evidence that the inhibition of both VACCs and transmitter release involves a PLC-dependent reduction in membrane PIP2. Materials and Methods Primary Cultures of Rat Superior Cervical Ganglion Neurons. Primary cultures of dissociated SCG neurons from neonatal rats were prepared as described previously (Boehm, 1999). Ganglia were dissected from 2to 6-day-old Sprague-Dawley rat pups that had been killed by decapitation in accordance with the rules of the university animal welfare committee. After incubation in collagenase (1.5 mg/ml; Sigma-Aldrich, Vienna, Austria) and dispase (3.0 mg/ml; Roche Diagnostics, Mannheim, Germany) for 45 min at 36°C, ganglia were trypsinized (0.25% trypsin; Worthington Biochemicals, Lakewood, NJ) for 20 min at 36°C, dissociated by trituration, and resuspended in Dulbecco’s modified Eagle’s medium (Invitrogen, Vienna, Austria) containing 2.2 g/l glucose, 10 mg/l insulin, 25,000 IU/l penicillin, 25 mg/l streptomycin (Invitrogen), 50 g/l nerve growth factor (R&D Systems, Minneapolis, MN), and 5% fetal calf serum (Invitrogen). Cells were plated either onto 5-mm discs (approximately 40,000 cells per disc) for [H]noradrenaline release or onto 35-mm culture dishes for electrophysiology. All tissue culture plastic was coated with rat tail collagen (Biomedical Technologies, Stoughton, MA). Cells were kept in a humidified 5% CO2 atmosphere at 36°C for up to 7 days, and one half of the medium was exchanged twice during this culture period. At 1 to 2 days before experiments, fresh medium without serum was added. Determination of [H]Noradrenaline Release. The release of [H]noradrenaline was determined as described before (Boehm, 1999). Cultures were labeled with 0.05 M [H]noradrenaline (specific activity, 71.7 Ci/mmol) in culture medium supplemented with 1 mM ascorbic acid at 36°C for 1 h. After labeling, culture discs were transferred to small chambers and superfused with a buffer containing 120 mM NaCl, 6.0 mM KCl, 2.0 mM CaCl2, 2.0 mM MgCl2, 20 mM glucose, 10 mM HEPES, 0.5 mM fumaric acid, 5.0 mM sodium pyruvate, 0.57 mM ascorbic acid, and 0.001 mM desipramine, adjusted to pH 7.4 with NaOH. Superfusion was performed at 25°C at a rate of approximately 1.0 ml/min. The collection of 4-min superfusate fractions was started after a 60-min washout period. Tritium overflow was evoked during two consecutive stimulation periods (S1 and S2) by the inclusion of either 0.3 mM ATP or 40 mM KCl (NaCl was reduced accordingly to maintain isotonicity) in the buffer for 60 s. Radioactivity released in response to electrical field stimulation from rat sympathetic neurons after labeling with tritiated noradrenaline and under conditions similar to those of the present study had been shown previously to consist predominantly of the authentic transmitter and to contain only small amounts ( 15%) of metabolites (Schwartz and Malik, 1993). Hence, the outflow of tritium measured in this study was assumed to reflect primarily the release of noradrenaline and not that of metabolites. Tetrodotoxin (TTX; 0.1 M), CdCl2 (100 M), and thapsigargin (0.3 M), if appropriate, were added to the superfusion buffer after 50 min of superfusion (i.e., 10 min before the start of sample collection). Bradykinin (1 M) and UK 14304 (1 M) were added to the superfusion buffer 2 min before and phenylarsine oxide (10 M) and dithiothreitol (1 mM) 4 min before the second stimulation period. At the end of experiments, the radioactivity remaining in the cells was extracted by immersion of the discs in 1.2 ml of 2% (v/v) perchloric acid followed by sonication. Radioactivity in extracts and collected fractions was determined by liquid scintillation counting (Packard Tri-Carb 2100 TR; PerkinElmer Life and Analytical Sciences, Bos-