The proportion of isolated rat dorsal root ganglion neurones responding to bradykinin increases with time in culture

Neuroscience Letters 252 (1998) 143–146 The proportion of isolated rat dorsal root ganglion neurones responding to bradykinin increases with time in culture M. Petersen*, A. Klusch, A. Eckert Department of Physiology, University of Würzburg, Röntgenring 9, D-97070 Würzburg, Germany Received 8 June 1998; received in revised form 7 July 1998; accepted 8 July 1998 Abstract The proportion of isolated rodent dorsal root ganglion neurones expressing bradykinin receptors increases transiently with time in culture. However, it has not yet been investigated whether these receptors are functioning. Therefore the responses of these neurones to bradykinin (1 mM) were investigated in patch-clamp experiments in the current clamp mode after 0.8 and 1.8 days under culture conditions. The proportion of neurones responding to bradykinin was 26% (5/19) at day 0.8 and increased to 73% (16/22) at day 1.8. The intensity of the response was assessed by counting the number of action potentials evoked by bradykinin within four fixed intervals of 500 ms duration during each experiment. It increased with time in culture from an average of 8 ± 2 (SD) at day 0.8 to 16 ± 6 at day 1.8, respectively. These results provide evidence for the induction of functioning bradykinin receptors in cultured dorsal root ganglion neurones with time in culture.  1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: Bradykinin; Patch-clamp; Dorsal root ganglion neurones; Cell culture; Hyperalgesia Several lines of evidence support a role for the endogenous nonapeptide bradykinin in hyperalgesia under inflammatory conditions [4]. In inflammatory exudate concentrations of bradykinin between 0.3 and 55 nM were measured [2,5]. However, the biological response to a ligand depends not only on the concentration present at the sensory terminal but also on the number of neurones expressing functional receptors and on the density of receptors per neurone. Bradykinin exerts its physiological effects by binding to specific receptors [6,8,13]. In rat and guinea-pig dorsal root ganglion (DRG) neurones, bradykinin receptors are constitutively expressed in a subpopulation of neurones [11,15, 16]. In previous studies we demonstrated a transient increase (i) in the proportion of DRG neurones expressing bradykinin receptors and (ii) in the proportion of neurones expressing a high density of bradykinin receptors with time in culture [15]. In addition, we found that under culture conditions the induction of bradykinin receptors strongly depends on the interaction of nerve growth factor (NGF) * Corresponding author. Tel.: +49 931 312722; fax: +49 931 312741 e-mail: marlen.petersen@mail.uni-wuerzburg.de with the low affinity neurotrophin receptor p75 [12]. However, from these experiments it is not clear whether de novo expressed receptors are also functioning. Therefore, in the study presented, the proportion of DRG neurones responding to bradykinin with action potentials at different points of time in culture was determined and compared with the proportion of neurones expressing bradykinin receptors at comparable points of time. Adult male Sprague–Dawley rats weighing 180–200 g were used. The isolation of DRG neurones was carried out as described previously [10,18]. Ganglia from all spinal levels were excised. The ganglia were incubated for 100 min at 37°C in collagenase type A (0.31 U/mg) and then for 11 min in trypsin (25 000 U/ml). Individual cells were obtained from ganglia by repeated trituration with a firepolished pipette. Cells were dispersed in Ham’s F-12 medium supplemented with 10% heat-inactivated horse serum, 2 mM l-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 100 ng/ml NGF 7S. Cells were plated on polyl-lysine coated coverslips and were maintained at 37°C in a humidified atmosphere of air gassed with 3.5% CO2. DRG neurones were current-clamped at the resting membrane potential by the whole-cell patch-clamp method with 0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00579- 5 144 M. Petersen et al. / Neuroscience Letters 252 (1998) 143–146 Fig. 1. Comparison between the proportion of neurones expressing bradykinin binding sites, detected by bradykinin-gold binding studies (white bars) and the proportion of neurones responding to bradykinin with action potentials, detected by patch-clamp experiments (black bars). Binding studies were done after 0.8 days (19 h) and 1.8 days (43 h) in culture (n = 3 cultures, 300 neurones for each time point); patch-clamp experiments were performed after 17–21 h and 41–45 h in culture (n = 6 cultures, 20–22 neurones for each time point), respectively, also labelled as 0.8 and 1.8 days. Data from binding studies are published in [15]. an Axopatch 200 A amplifier (Axon Instruments). Cell capacitance was compensated by nulling circuitry in the amplifier. The clamp command signals were generated via the amplifier and a PC with a DigiData 1200 interface and pClamp 6.0 software (Axon Instruments). The response of neurones to bradykinin (1 mM) was tested in the currentclamp mode. Because bradykinin alone often does not depolarize the membrane to the threshold that is required to generate action potentials, membrane depolarization steps for generating action potentials were induced according to Jones et al. [7]. Before bradykinin application the threshold membrane potential for generating action potentials was determined by injection of a current step of 0.3, 0.5 or 1.0 nA for 500 ms. Bradykinin was then applied for 2–4 min. During this period, depolarizing current injections were performed every 30 s for 500 ms. The first action potential during each depolarization step is evoked by the current injection itself, the subsequent ones by bradykinin (see Fig. 2A–C). Recordings were done at a sampling rate of 4 kHz. Electrodes were fire-polished and had a final resistance that ranged between 2 and 6 MQ. They were filled with (mM): KCl 140, CaCl2 1, EGTA 11, HEPES 10, Mg-ATP 2, adjusted with KOH to a pH of 7.3. The normal external solution consisted of (mM): NaCl 140, KCl 5, CaCl2 2, MgCl2 1 and was adjusted with NaOH to a pH of 7.3. A coverslip with the cells was placed in a recording chamber, which contained approximately 0.5 ml of the external solution. The cells were continuously superfused with the normal external solution or the bradykinin containing solution, respectively, by a flow/suction device. The flow rate was about 7 ml/min. The experiments were performed at room temperature. The cross sectional area of each soma was calculated in mm2 from the formula (a/2 × b/2) × p, where a is the major and b the minor cell axis. In studies using gold-labelled bradykinin, 43 ± 7% (SEM) of DRG neurones expressed bradykinin receptors at day 0.8 in culture. This proportion increased to 85 ± 8% at day 1.8 [15]. To test whether the induced bradykinin receptors are not only incorporated into the membrane, but are also functioning, electrophysiological recordings on isolated neurones were performed during time windows of 17 to 21 h and 41 to 45 h in culture, referred to as 0.8 and 1.8 days in culture (Fig. 1). In a subpopulation of neurones bradykinin evoked action-potentials during injection of depolarizing current steps (Fig. 2BC). After 0.8 days in culture 26% (5/19) of neurones responded to 1 mM bradykinin with action potentials and after 1.8 days the proportion was 73% (16/22) (Fig. 1). A chi2 test indicated a significant increase of neurones after 1.8 days in culture which gener- Fig. 2. Responses of three different neurones to bradykinin (1 mM) during depolarizing current steps of 500 ms duration, respectively. The first action potential in each recording is caused by the current injection. A: bradykinin-unresponsive neurone. B,C: bradykininresponsive neurones. The resting membrane potential was −58 mV (A), −58 mV (B) and −57 mV (C) after 0.8 days (A,B) and 1.8 days (C) in culture, respectively. The cross sectional area was 630 mm2 (A), 690 mm2 (B) and 729 mm2 (C), respectively. M. Petersen et al. / Neuroscience Letters 252 (1998) 143–146 ated action potentials in response to bradykinin compared to neurones after 0.8 days (P , 0.01). In our previous bradykinin-gold binding studies there was not only an increase in the proportion of neurones expressing bradykinin receptors but also an increase in the proportion of neurones expressing a high density of these receptors [15]. To test whether the increased number of receptors in individual neurones leads to an increase in the generation of action potentials, the number of action potentials evoked by bradykinin was compared between neurones which had been under culture conditions for 0.8 and 1.8 days, respectively. To this end, the number of action potentials evoked by bradykinin during the first four current injections of 500 ms each was counted. The current injections were done after onset of bradykinin application and then after every 30 s. In the accumulated 2.0 s time period, the average number of action potentials evoked by 1 mM bradykinin was 8 ± 2 at day 0.8 and increased to 16 ± 6 at day 1.8. The maximum response of a neurone at day 0.8 in culture was 11 action potentials and at day 1.8 it was 27 action potentials during the 2.0 s test period. Three different neurones responding differently to 1 mM bradykinin are shown in Fig. 2. The first action potential in each recording is evoked by the current injection. In Fig. 2A, an example of a bradykinin unresponsive neurone is shown. In Fig. 2B bradykinin evoked one action potential and subthreshold oscillations of the membrane potential, whereas the neurone in Fig. 2C showed repetitive firing of action potentials. The neurones shown in Fig. 2A,B had been under culture conditions for 0.8 days, the one in Fig. 2C for 1.8 days. At the two points of time in culture the resting membrane potential as well as the cross sectional area were in a comparable range. At day 0.8 the potential was −58 ± 2 mV (SEM) and at day 1.8 it was −52 ± 2 mV. The cross-sectional area of responsive neurones was between 596 and 973 mm2 at day 0.8 and between 335 and 1530 mm2 at day 1.8 (Fig. 3). The data presented show that under culture conditions there is an increase in the number of DRG neurones responding to bradykinin between 0.8 and 1.8 days in culture, as well as an increase in the frequency of action potentials generated by bradykinin, indicating that mechanisms regulate the sensitivity of DRG neurones to bradykinin during culture. These data correspond well with our previous data of gold-labelled bradykinin receptors, which demonstrate a significant increase in the proportion of neurones with these receptors at day 1.8 in culture compared to day 0.8 (see Fig. 1) as well as an increase in the proportion of neurones with a higher density of receptors [11,12,15]. The somewhat lower percentage of 26% found in the electrophysiological studies at day 0.8 could be due to the lower densities of bradykinin receptors at that point in time. They may not have been sufficient for generating action potentials. In the binding studies we found a de novo expression of the B1 receptor subtype and an up-regulation of the B2 145 Fig. 3. Distribution of cross sectional area of bradykinin (1 mM) responsive neurones (black bars) and bradykinin unresponsive neurones (white bars). Recordings were done 0.8 days (17–21 h; top) and 1.8 days (41–45 h; bottom) after isolation of neurones. Bin width is 50 mm2. Same experiments as shown in Fig. 1. subtype [15]. Therefore, we assume that both subtypes are also involved in the increase in functional receptors. In a recent paper we also demonstrated that the up-regulation of bradykinin receptors strongly depends on the presence of NGF, however, NGF does not affect their basal expression [12]. In the experiments presented, DRG neurones were cultured in the presence of NGF; therefore, one can assume that the increase in responsiveness to bradykinin is regulated via NGF-dependent induction of bradykinin receptors. A recruitment of isolated DRG neurones responding to bradykinin has also been shown following treatment with PGE2, demonstrated by an increase of the intracellular calcium concentration [17]. An increase in the concentration of NGF in inflamed tissue in vivo has been shown [1,3,9]. Behavioural experiments in rats suggest that this increase causes an induction of bradykinin receptors [14], but the cell type involved remained unclear. Our findings under in vitro conditions show an induction of functioning bradykinin receptors in sensory neurones. This induction occurred both in neurones which expressed bradykinin receptors constitutively and in those without constitutive receptors [11,15]. Under pathophysiological conditions in vivo, the induction of these receptors could result in increased responsiveness of primary sensory neurones and could promote chronic pain 146 M. Petersen et al. / Neuroscience Letters 252 (1998) 143–146 states. As many of the primary afferent neurones respond not only to the algesic substance bradykinin, but also to other stimuli like thermal and mechanical ones, the increase of functional bradykinin receptors could contribute to sensitization of these sensory neurones via membrane depolarization when simultaneously activated by other stimuli. The demonstration of an increase in the proportion of neurones expressing functioning bradykinin receptors in vitro further suggests a contribution of bradykinin to pain and hyperalgesia by spatial summation in vivo. In addition, an increased understanding of the mechanisms of plasticity of the expression of bradykinin receptors may lead to improved therapy for chronic pain. 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