The Journal of Neuroscience, July 15, 2002, 22(14):5823–5832 Sympathoexcitation by Bradykinin Involves Ca2؉-Independent Protein Kinase C Thomas Scholze, Eugenia Moskvina, Martina Mayer, Herwig Just, Helmut Kubista, and Stefan Boehm Department of Pharmacology, University of Vienna, A-1090 Vienna, Austria Bradykinin has long been known to excite sympathetic neurons the neurons, but left the cellular distribution of other isoforms 2ϩ via B2 receptors, and this action is believed to be mediated by unchanged. This activation of Ca -independent protein kinase C an inhibition of M-currents via phospholipase C and inositol enzymes was prevented by a phospholipase C inhibitor. The trisphosphate-dependent increases in intracellular Ca 2ϩ.Inpri- bradykinin-dependent stimulation of noradrenaline release was mary cultures of rat superior cervical ganglion neurons, bradykinin reduced by inhibitors of classical and novel protein kinase C caused an accumulation of inositol trisphosphate, an inhibition of isozymes, but not by an inhibitor selective for Ca 2ϩ-dependent M-currents, and a stimulation of action potential-mediated trans- isoforms. These results demonstrate that bradykinin B2 receptors mitter release. Blockade of inositol trisphosphate-dependent sig- are linked to phospholipase C to simultaneously activate two naling cascades failed to affect the bradykinin-induced release of signaling pathways: one mediates an inositol trisphosphate- and noradrenaline, but prevented the peptide-induced inhibition of Ca 2ϩ-dependent inhibition of M-currents, the other one leads to M-currents. In contrast, inhibition or downregulation of protein an excitation of sympathetic neurons independently of changes in kinase C reduced the stimulation of transmitter release, but not the M-currents through an activation of Ca 2ϩ-insensitive protein ki- inhibition of M-currents, by bradykinin. In cultures of superior nase C. cervical ganglia, classical (␣, ␤I, ␤II), novel (␦, ⑀), and atypical (␨) protein kinase C isozymes were detected by immunoblotting. Key words: rat superior cervical ganglion neurons; noradren- Bradykinin induced a translocation of Ca 2ϩ-independent protein aline release; bradykinin; M-type K ϩ channels; protein kinase C; kinase C isoforms (␦ and ⑀) from the cytosol to the membrane of phospholipase C Bradykinin is formed through the actions of kallikreins from potentials and become completely activated when neurons are plasma kininogens in response to various noxious stimuli. The further depolarized. Hence, activated KM channels keep neurons nonapeptide acts as a potent mediator of inflammation and pain polarized, and closure of these ion channels causes depolariza- through its effects on peripheral sensory neurons. On one hand, tion and increased action potential discharge (Brown, 1983; Mar- the stimulation of nociceptive nerve terminals elicits the painful rion, 1997). Recently, the signaling cascade mediating the inhibi- sensation, on the other hand, bradykinin induces the release of tion of KM channels by bradykinin has also been elucidated: B2 neuropeptides that then contribute to the inflammatory response bradykinin receptors are linked to G-proteins of the Gq family, (Bhoola et al., 1992). Nevertheless, the sympathetic nervous sys- ␣ most likely G11, and it is the subunits that mediate the closure tem may also contribute to these actions because bradykinin- of KM channels (Jones et al., 1995; Haley et al., 1998, 2000a). Via induced hyperalgesia (Levine et al., 1986) and plasma extravasa- these G-proteins, phospholipase C-␤4 is stimulated (Haley et al., tion (Miao et al., 1996) are abolished after sympathectomy. In 2000b) to mediate the synthesis of inositol trisphosphate (IP3), ϩ addition, the sympathetic nervous system may also be involved in which then causes liberation of Ca 2 from intracellular stores both the protective as well as the noxious effects that bradykinin 2ϩ (Cruzblanca et al., 1998). Finally, cytosolic Ca concentrations has been suggested to exert within the cardiovascular system in the submicromolar to low micromolar range directly block K (Dell’Italia and Oparil, 1999). M channels (Selyanko and Brown, 1996). Almost 40 years ago, bradykinin was found to cause sympatho- Apart from inhibiting K channels, bradykinin was shown to excitation attributable to its depolarizing action on sympathetic M either enhance (Cox et al., 2000) or reduce (Starke et al., 1977) ganglia (Lewis and Reit, 1965; Trendelenburg, 1966). For three decades, the mechanisms underlying this effect remained un- sympathetic transmitter release via presynaptic sites of action, known, but then Jones et al. (1995) demonstrated that the peptide depending on the species and tissues investigated. In primary depolarized superior cervical ganglion (SCG) neurons of the rat cultures of rat SCG neurons, activation of B2 bradykinin recep- ϩ tors causes action potential-dependent transmitter release through an inhibition of M-type K (KM) channels. These ion (Boehm and Huck, 1997). This effect is selectively potentiated by channels are opened in the subthreshold voltage range for action ϩ KM channel blockers, but not by blockers of other types of K Received Feb. 25, 2002; revised April 9, 2002; accepted April 12, 2002. channels. In addition, KM channel blockers were found to stimu- The study was supported by the “Virologiefonds” of the University of Vienna late transmitter release from sympathetic neurons in the absence and the Austrian Science Fund (Fonds zur Fo¯rderung der Wissenschaftlichen Forschung; Grant P13920-MED). We are indebted to M. Freissmuth for valuable of any other secretagogue stimulus (Kristufek et al., 1999). There- comments on this manuscript. fore, it appeared obvious to assume that peptide-induced nor- Correspondence should be addressed to Stefan Boehm, Department of Pharma- adrenaline release was also mediated by the signaling cascade that cology, University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria. E-mail: [email protected]. finally leads to KM channel blockade. However, the present results Copyright © 2002 Society for Neuroscience 0270-6474/02/225823-10$15.00/0 demonstrate that bradykinin also uses another signaling cascade 5824 J. Neurosci., July 15, 2002, 22(14):5823–5832 Scholze et al. • Bradykinin and Protein Kinase C ϩ to excite sympathetic neurons, which includes Ca 2 -independent with1mM ascorbic acid at 36°C for 1 hr. After labeling, culture discs were transferred to small chambers and superfused with a buffer containing protein kinase C (PKC), but bypasses KM channels. (in mM): NaCl (120), KCl (6.0), CaCl2 (2.0), MgCl2 (2.0), glucose (20), MATERIALS AND METHODS HEPES (10), fumaric acid (0.5), Na-pyruvate (5.0), ascorbic acid (0.57), and desipramine (0.001), adjusted to pH 7.4 with NaOH. Superfusion Primary cultures of rat superior cervical ganglion neurons. Primary cultures was performed at 25°C at a rate of ϳ1.0 ml/min. Collection of 4 min of dissociated SCG neurons from neonatal rats were prepared as de- superfusate fractions was started after a 60 min washout period to scribed before (Boehm, 1999). Briefly, ganglia were dissected from 2- to remove excess radioactivity. 6-d-old Sprague Dawley rat pups, cut into three or four pieces and To investigate the mechanisms of bradykinin-evoked noradrenaline incubated in collagenase (1.5 mg/ml; catalog #9891; Sigma, Vienna, release, [ 3H] overflow was induced by the inclusion of the peptide in the Austria) and dispase (3.0 mg/ml; catalog #165859; Boehringer Mann- superfusion buffer for 2 min, unless indicated otherwise. Cell-permeable heim, Mannheim, Germany) for 20 min at 36°C. Subsequently, the phorbol esters, in contrast, were included for periods of 12 min to ganglia were trypsinized (0.25% trypsin; catalog #3703; Worthington, 3 stimulate [ H] overflow. Modulatory agents, such as U73122, U73343, Freehold, NJ) for 15 min at 36°C, dissociated by trituration, and resus- thapsigargin, or diverse protein kinase C inhibitors were added to the pended in DMEM (catalog #041–01885M; Invitrogen, Gaithersburg, buffer after 50 min of superfusion (i.e., 10 min before the start of sample MD) containing 2.2 gm/l glucose, 10 mg/l insulin, 25000 IU/l penicillin, collection). The buffer then remained unchanged until the end of exper- and 25 mg/l streptomycin (catalog #043–05140D; Invitrogen), 10 ␮g/l iments. To control for unspecific effects of these modulatory agents, nerve growth factor (catalog #0436050; Invitrogen), and 5% fetal calf tritium overflow was also elicited by the application of 36 monophasic serum (catalog #011–0620H; Invitrogen). Thereafter, the cells were rectangular electrical pulses (0.5 msec, 50 mA, 50 V/cm) at 0.3 Hz. At the plated onto 5 mm discs for superfusion experiments and onto 35 mm end of experiments, the radioactivity remaining in the cells was extracted culture dishes (catalog #153066; Nunc, Naperville, IL) for electrophys- by immersion of the discs in 2% (v/v) perchloric acid followed by iological experiments, both coated with rat tail collagen (Biomedical sonication. Radioactivity in extracts and collected fractions was deter- Technologies, Stoughton, MA). For the determination of cellular inositol mined by liquid scintillation counting (Packard Tri-Carb 2100 TR). phosphates, cells were plated onto 24 multiwell plates (Nunc; 200,000 Radioactivity released in response to electrical field stimulation from rat cells per well) coated with poly-D-lysine (Sigma). All cultures were kept sympathetic neurons after labeling with tritiated noradrenaline under