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The Journal of Physiology logoLink to The Journal of Physiology
. 2004 May 28;558(Pt 2):513–526. doi: 10.1113/jphysiol.2004.066381

Bradykinin decreases K+ and increases Cl− conductances in vagal afferent neurones of the guinea pig

Eun Joo Oh 1, Daniel Weinreich 1
PMCID: PMC1664971  PMID: 15169850

Abstract

Bradykinin (BK) is an inflammatory mediator that can excite and sensitize primary afferent neurones. The nature of the ionic channels underlying the excitatory actions of BK is still incompletely understood. Using whole-cell patch-clamp recording from acutely dissociated nodose ganglion neurones (NGNs) we have examined the ionic mechanism responsible for BK's excitatory effect. Bath-applied BK (0.1 μm) depolarized the membrane potential (29 ± 3.1 mV, n = 7), evoked action potentials, and induced an inward ionic current (IBK) with two distinctive membrane conductances (gm). Initially, gm decreased; the ionic current associated with this gm had a reversal potential (Erev) value of −87 ± 1.1 mV (n = 26), a value close to EK (−89 mV). Subsequently, gm increased; the ionic current associated with this gm had an estimated Erev of 49 ± 4.3 mV (n = 23). When the second component was isolated from the first component, by replacing [K+]o with Cs+, Erev was 20 ± 4.7 mV (n = 10). Replacing external NaCl with NMDG-Cl or choline-Cl, or reducing [Ca2+]o did not significantly diminish IBK. After replacing external NaCl with sodium isethionate, Erev for the second component shifted to 56 ± 8.8 mV (n = 4), a value close to the ECl (66 mV). The second component was inhibited by intracellular BAPTA or by bath application of niflumic acid (100 μm), a Ca2+-activated Cl− channel blocker. These results suggest that the first and second components of IBK are produced by a decrease in K+ conductance and an increase in Ca2+-activated Cl− conductance, respectively. The BK-evoked Cl− conductance in NGNs may be the first demonstration of an inflammatory mediator exciting primary afferents via an anion channel.

The nonapeptide bradykinin (BK) is a potent inflammatory mediator synthesized from its precursor, high molecular weight kininogen, by activated tissue and plasma kallikreins. BK can act on non-neuronal as well as neuronal tissue to produce a wide range of effects including hypotension, smooth muscle contraction, vasodilatation and pain (Proud & Kaplan, 1988; Dray & Perkins, 1993). In neuronal tissues, BK receptors are mainly localized to nociceptive primary afferent neurones (Steranka et al. 1988) including visceral and somatic afferents. Upon activation, BK receptors can increase the excitability of sensory neurones through diverse mechanisms (Wood & Docherty, 1997). In the airways, BK can excite vagal afferent neurones initiating reflexes such as bronchoconstriction and mucus secretion promoting airway obstruction (Ichinose et al. 1990; Kajekar et al. 1999). The afferent limb of these reflexes is mediated by sensory fibres whose cell bodies reside in the nodose ganglia (NGs) and jugular ganglia (JGs). The mechanism by which BK excites airway primary afferents in NGs and JGs has not yet been clearly established.

Several ion channels have been identified as final effectors following BK receptor activation. In neuroblastoma–glioma hybrid cell line, BK application produces biphasic responses – a transient outward current with increased membrane conductance (gm) followed by an inward current with decreased membrane conductance. These currents were attributed to activation of a Ca2+-activated K+ current and an inhibition of an M-current, respectively (Higashida & Brown, 1986). In the somata of a restricted population of C-type NG neurones (NGNs), BK can abolish spike frequency accommodation by blocking a Ca2+-activated K+ current associated with a slow afterhyperpolarization (Weinreich, 1986; Weinreich & Wonderlin, 1987). This sensitizing action of BK was mediated by the formation of prostacyclin subsequent to the activation of B2 receptors (Weinreich et al. 1995). In isolated neonatal DRG neurones (DRGNs), BK evokes an inward current and an increased gm attributed to the opening of Na+ channels in a protein kinase C-dependent manner (Burgess et al. 1989). BK can modify TRPV1 receptors (vanilloid receptor 1), a non-specific cation channel, through a lipid signalling pathway. In neonatal DRGNs, activation of BK receptors leads to the generation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), a lipoxygenase product that can act as an agonist at TRPV1 receptors (Shin et al. 2002).

We have reported that BK can induce inward currents associated with an increased and a decreased gm in acutely isolated spinal primary afferent neurones (DRGNs) innervating the airway of adult guinea pigs (Oh et al. 2003). The present work was undertaken to determine the ionic mechanism responsible to these effects of BK in vagal afferent neurones (NGNs). Our results reveal that the increased gm evoked by BK is due to a Ca2+-activated Cl− conductance activated by a B2 receptor while the decrease in the gm component is due to a decrease in K+ conductance.

Methods

Retrograde labelling of airway specific sensory neurones

Male Hartley guinea pigs (200–250 g, Charles River, Wilmington, MA, USA) were anaesthetized with 60 mg kg−1 ketamine−10 mg kg−1 xylazine (i.p.). The lipophilic retrograde tracer DiI (Molecular Probes, Eugene, OR, USA) was prepared to a final concentration of 0.5 mg ml−1 in 1 % ethanol. The middle cervical trachea was exposed with midline incision in the neck and 400 μl of DiI solution was instilled to the lumen of the airway using a 28.5-gauge needle. Animals were positioned with their heads elevated throughout surgical procedures and recovery. Animals were closely observed for breathing patterns until they woke up from anaesthesia (∼20 min). After a period of 10–12 days guinea pigs were killed by an overdose (0.1 g kg−1, i.p.) of pentobarbital sodium as approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore. Their nodose ganglia (NGs) were removed and prepared for dissociation. These time periods were chosen to allow retrograde transport of the dye from the airway to the NGs and to allow recovery from any inflammatory responses that might be provoked by surgery. NGs from 200 to 300 g non-labelled animals were also used for this study.

Dissociation and acute culture of neurones

Nodose ganglion neurones (NGNs) were dissociated enzymatically and mechanically. After connective tissues and vessels were carefully removed from NGs, ganglia were incubated in an enzyme solution containing 5 mg collagenase type IA (Sigma, St Louis, MO, USA) and 5 mg dispase II (Boehringer Mannheim, Mannheim, Germany) in 5 ml of Ca2+- and Mg2+-free Hanks' balanced salt solution for 2 h 15 min at 37°C. NGNs were dissociated by trituration with Pasteur pipettes of decreasing tip diameters. Enzyme solutions were replaced with culture medium containing L15 medium (Gibco BRL, Rockville, MD, USA) and 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA) by centrifugation (3 times at 700 g for 45 s) then re-suspended with culture medium. Culture medium (150 μl) containing dissociated neurones was transferred to circular 25 mm glass coverslips (Fisher, Newark, DE, USA) coated with poly-d-lysine (0.1 mg ml−1). Two hours after plating, 2 ml of additional culture medium was added to the culture dishes. Neurones were maintained in culture at 37°C prior to recording. Electrophysiological data were obtained 2–9 h after dissociation.

Electrophysiology

Whole-cell patch-clamp recording techniques were employed with an Axopatch 200B amplifier and pCLAMP8 software (Axon Instruments, Union City, CA, USA). Patch pipettes with resistance 1–3 MΩ were fabricated from glass capillaries (MTW150F-4, World Precision Instruments, Sarasota, FL, USA). Pipettes were filled with a solution containing (mm): 135 KCl, 10 NaCl, 2 MgCl2, 1 CaCl2, 10 N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid] (Hepes), 11 ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 2 Mg-ATP, and 1 Li-GTP; pH 7.3 adjusted with KOH, 324 mosmol l−1. After pH correction, [K+]i was 165 mm. For Cs+-based intracellular solution, 130 mm CsCl was substituted for 135 mm KCl and pH was corrected with CsOH. In some experiments, EGTA (11 mm), was replaced by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA, 10 mm) for faster chelation of Ca2+. During recording, coverslips were continuously superfused (6–7 ml min−1) with Locke solution or Hepes-buffered physiological salt solution maintained at 33°C. Bicarbonate-buffered Locke solution had the following composition (mm): 136 NaCl, 5.6 KCl, 1.2 NaH2PO4, 14.3 NaHCO3, 1.2 MgCl2, 2.2 CaCl2, and 10 dextrose, equilibrated with 95% O2–5% CO2, pH 7.3–7.5. Hepes-buffered physiological salt solution contained (mm): 150 NaCl, 5.6 KCl, 1.2 MgCl2, 2.2 CaCl2, 10 dextrose, 10 Hepes, pH 7.4 adjusted with NaOH. An equimolar concentration of N-methyl-d-glucamine chloride (NMDG-Cl) or choline-Cl was used for NaCl replacement. Ca2+ and Cl− were replaced by Mg2+ and isethionate, respectively. Pipette voltage offset was neutralized prior to the formation of a gigaohm seal. Membrane resistance (Rm), series resistance (Rs) and membrane capacitance (Cm) were determined from current transients elicited by a 5 mV depolarizing step from a holding potential of −60 mV, using the ‘Membrane Test’ application of pCLAMP8. Capacitance and 80% Rs were compensated. Criteria for cell inclusion in the study were as follows: Rs ≤ 5 MΩ, Rm > 100 MΩ, and stable recording with 80% Rs compensation throughout the experiment. Once neurones were stabilized (usually 2–3 min), 3 ms depolarizing current pulses were applied in current-clamp mode to generate action potentials. We were able to exclude recording from glia (satellite cells) by their low Rm and absence of measurable action potentials. BK was bath-applied for 30 s at a concentration of 0.1 μm, except when studying airway-identified NGNs where 1 μm was used. For monitoring membrane conductance (gm) and estimating reversal potential (Erev) values, ramp voltage commands were applied repetitively (from −110 to −50 mV, 0.2 mV ms−1, 2 Hz or 0.3 mV ms−1, 1 Hz). gm was estimated by the slope of the ramp current. The recording chamber was grounded via a 3 m KCl agar bridge. Junction potentials were calculated using pCLAMP 8 and corrected.

Chemicals

BK was purchased from Calbiochem (San Diego, CA, USA) and stored in 1 mm aliquots at −20°C. Iodo-resiniferatoxin (iRTX) and 5,8,11,14-eicosatetraynoic acid (ETYA) were gifts from Dr Bradley J. Undem. Salts and dextrose were purchased from J. T. Baker (Phillipsburg, NJ, USA) and all the other chemicals were from Sigma.

Data analysis

Data obtained from pCLAMP8 software were analysed and plotted using Clampfit8 software (Axon Instruments) and SigmaPlot 2000 (SPSS, Chicago, IL, USA). Statistical tests were performed with SigmaStat 2.0 (SPSS) and values were presented as means ± s.e.m. P < 0.05 represented statistical significance.

Results

Effects of BK on NGNs innervating airway

Seven per cent (7 ± 0.3%) of the isolated somata in NG were labelled with DiI. This value was obtained from three independent dissociations of three animals; in each dissociation, 5–7 fields of each coverslip were examined at 40×; at least three coverslips were examined in each dissociation. In about 40% (10/25) of nodose ganglion neurones (NGNs) retrogradely dye-labelled from the airway, BK (1 μm) evoked inward currents ranging between 0.1 and 1.3 nA. We measured the effect of BK on the membrane conductance (gm) in four airway-identified NGNs using ramp voltage commands (−110 to −50 mV, 0.2 mV ms−1, 2 Hz). In all four NGNs, BK produced biphasic changes in gm associated with the inward currents; during the initial phase of the response there was a decrease in gm then a larger and more sustained increase in gm. The traces in Fig. 1 illustrate the time course of the BK-evoked inward current and the changes in gm during this response. A reversal potential (Erev) value was estimated for each component by extrapolating ramp currents induced by ramp voltage commands before BK treatment and at various times during the BK response (see Figs 1, 2 and 3). The estimated Erev value for the BK response associated with a decreased gm was −86 ± 1.9 mV (n= 4), a value close to equilibrium potential for K+ (EK= −89 mV) predicted by the ionic conditions used (see Methods). During the peak of the responses, when there was a profound increase in gm, the estimated Erev was 44 ± 4.5 mV (n= 3). In one of the four NGNs, the control and BK ramp currents nearly paralleled one another, prohibiting the determination of an Erev value.

Figure 1. BK-induced response in airway-identified NGNs and effects of BK on the membrane potential of NGNs.

Figure 1

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A, BK (1 μm) induced inward current in NGN innervating airway. This current was mainly related to the increased membrane conductance (gm) but, at the beginning of the inward current, there was transient decrease in gm. Asterisks indicate three time points where gm changes. B, ramp currents generated by repetitive ramp voltage commands were overlaid at three time points indicated by asterisks in A. Initially, gm decreased, indicated by decreased slope of ramp current. At the peak of the response, large inward current accompanied with increased gm, indicated by increased slope of ramp current. The ramp voltage command (from −110 to −50 mV, 300 ms) is displayed in the lowest panel. C, the membrane potential of NGNs depolarized upon bath application of 0.1 μm BK. This depolarization led to action potential discharges, indicating excitatory effect of BK on NGNs. This particular NGN had resting membrane potential −54 mV. Inset, an extracellular recording made with a patch pipette in an on-cell recording mode showing action potential discharges upon BK application. Horizontal bars depict the time of BK application.

Figure 2. Representative biphasic response to BK application and the changes of membrane conductance (gm) and Erev during the response in a NGN.

Figure 2

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Bath application of BK (0.1 μm, 30 s) at 33°C induced inward current with two distinctive gm changes. Initially, BK induced a transient inward current with decreased gm (point a). As the inward current progressed further, gm increased and this was completely counterbalanced by a decreased gm, resulting in almost parallel I–V plots (point b). At the peak of the response (point c), gm was apparently increased compared to control (baseline). Insets were I–V plots from ramp currents induced by repetitive ramp voltage commands. Control indicates a baseline ramp current obtained before the BK application and BK indicates a ramp current at three different time points a, b and c. The horizontal bar depicts the time of BK application. B, the time courses of changes in gm and the Erev values from the above response were plotted. Baseline gm was 8.1 nS and it decreased to 6.2 nS at point a. The Erev value at this point was −99 mV, indicating inhibition of a K+ conductance. Then, gm slowly increased and at point b it was 7.8 nS, which is similar to baseline gm. At this point, Erev was not measurable (out of range) because I–V plots were almost parallel with time At the peak of the response gm was 14.4 nS, point c. An Erev value of 62 mV at this point suggested the opening of cation channel(s) such as Na+ and/or Ca2+ channels.

Figure 3. Representative monophasic responses upon BK application.

Figure 3

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A, in a few NGNs, BK (0.1 μm) induced inward currents related to decrease in gm only. These responses were smaller in current amplitude and reached the peak faster than the responses related to increased gm. In this particular neurone, the current amplitude was 0.1 nA and it reached the peak at 15 s. The Erev value for this response was −90 mV (inset), which is consistent with the initial component of biphasic responses. B, BK evoked inward currents related to increase in gm only in some of NGNs. This particular response had an Erev value of 24 mV, which is a less depolarizing membrane potential compared to the Erev value in the second component of biphasic BK responses. This increased the possibility that the Erev value of the increased gm component in the biphasic BK response might be affected by an underlying decreased gm component persistent at that time point, leading to further depolarizing Erev values. The peak amplitude of this response was 0.5 nA and it took about 31 s to reach the peak from the initiation of response. Horizontal bars depict the time of BK application.

Sensitivity of unidentified NGNs to BK

We performed further electrophysiological studies in NGNs without identification of their target organs because: (1) only a limited number of airway sensory neurones were responsive to BK (∼7% of dissociated NGNs were airway-identified and of these ∼40% were responsive to BK), and (2) BK-evoked responses desensitized dramatically, limiting further studies in the same neurones. In an initial survey of unidentified NGNs, 34% (20/58) of the neurones sampled showed an inward current upon BK (0.1 μm) application. Further study revealed that the response to BK was limited to small- to medium-sized neurones (<35 μm). The response rate increased to 76% (41/54) by selecting this subpopulation of NGNs. BK-responsive NGNs had diameters of 29 ± 0.5 μm (n = 59) and Cm of 31 ± 1.2 pF (n = 61). Thus, in the work described below we used unidentified NGNs to study the ionic mechanisms and pharmacological properties of the biphasic BK responses.

Effect of BK on neuronal excitability

We first evaluated the actions of BK on unidentified NGNs under current-clamp recording conditions. Bath-applied BK (0.1 μm) evoked membrane depolarizations that averaged 29 ± 3.1 mV (n= 7, range 22–42 mV) and were accompanied by bursts of action potentials (Fig. 1C). Since whole-cell voltage-clamp recording disturbs intracellular ionic compositions, we applied BK while recording in the on-cell mode prior to membrane rupture. Under this recording condition, BK induced action potential discharges (Fig. 1C inset, n= 2). These results show that BK can exert powerful excitatory effects in NGNs.

Two distinctive components of BK-induced inward currents

As observed with airway-identified NGNs, biphasic gm changes were also recorded in unidentified NGNs. Seventy-two per cent of the BK-responsive NGNs showed an early decrease in gm followed by a protracted increase in gm (Table 1). The estimated Erev values for the currents associated with the decreased and increased gm were −87 ± 1.1 mV (n= 26) and 49 ± 4.3 mV (n= 23), respectively. We were unable to estimate Erev values for increased membrane conductance in nine NGNs because the I–V plots were almost parallel and their intersection would occur beyond the biological range. This observation might indicate that the decreased gm component was counterbalancing the increased gm component. Data supporting this inference are shown in Fig. 2A. At first, gm decreased, during the initial phase of the inward current, indicated by decreased slope conductance of the I–V plot (Fig. 2A, point a). As inward current progressed, slope conductance formed parallel lines, suggesting no net gm changes due to a balance between decreased and increased gm (Fig. 2A, point b). Further on during the inward current and at the peak of the response, a clear increase in gm was observed, indicated by the increased slope conductance in I–V plot (Fig. 2A, point c). When gm was monitored every 2 s during the response, a biphasic change in gm was observed (Fig. 2B, upper panel). gm decreased rapidly at first, then recovered to baseline conductance. Subsequently, gm increased slowly reaching a peak before slowly subsiding. The changes of Erev were also monitored together with gm changes (Fig. 2B, lower panel). At the point where gm was decreased, Erev was −99 mV, suggesting the closing of K+ channel upon activation of BK receptors. At the peak of the response, where gm was maximal, Erev was 62 mV, suggesting the opening of cationic channel(s) such as Na+ and Ca2+. Between these two time periods, there was little net gm change and Erev values were not measurable. These data further support the presence of two components in BK-induced inward currents in NGNs with distinctive ionic mechanisms.

Table 1.

Characteristics of BK-induced inward currents in varying intracellular solution

Intracellular solution Conductance % of BK(+) neurones Peak amplitude (nA) Erev (mV)
135 mm [KCl]i, 11 mm [EGTA]i Decrease only 6% (3/54) 0.1 ± 0.04*(n = 3) −89 ± 0.5 (n = 2)
Increase only 22% (12/54) 1.4 ± 0.48*(n = 12) 24 ± 2.6 (n = 12)
Decrease and increase 72% (39/54) 0.7 ± 0.09* (n = 39) 49 ± 4.3 (n = 23)
130 mm [CsCl]I, 11 mm [EGTA]I Decrease only 0% (0/12) — —
Increase only 92% (11/12) 0.3 ± 0.07 (n = 10) 20 ± 4.7 (n = 10)
Decrease and increase 8% (1/12) 0.3 (n = 1) 36 (n = 1)
135 mm [KCl]i, 10 mm [BAPTA]i Decrease only 86% (6/7) 0.1 ± 0.02 (n = 6) −87 ± 3.6 (n = 6)
Increase only 0% (0/7) — —
Decrease and increase 14% (1/7) 0.5 (n = 1) 53 (n = 1)
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In the condition of [KCl]i/[EGTA]i, [CsCl]i/[EGTA]i, and [KCl]i/[BAPTA]i, responsiveness to BK was tested in 112 NGNs from 20 animals, 20 NGNs from 2 animals, and 8 NGNs from 1 animal, repectively. Numbers in parenthesis indicate the number of NGNs with corresponding conductance change out of BK-responsive NGNs. Values in peak amplitude and Erev are means ± s.e.m.

*

P = 0.007 with Kruskal-Wallis one-way ANOVA on rank test.

A few NGNs showed only a single type of gm change during the BK-evoked inward current (Table 1). Six per cent of the BK responses were associated with a single gm change – a decrease in gm (Table 1 and Fig. 3A). The maximal amplitude of these responses was smaller (0.2 ± 0.04 nA, n = 3) than those observed during the peak of the biphasic response (0.7 ± 0.09 nA, n = 38) or the monophasic increase gm response (1.4 ± 0.48 nA, n = 12; see below). These values were significantly different (P = 0.007, Kruskal-Wallis one-way ANOVA on rank). Nonetheless, their Erev values (−88 and −90 mV; n = 2) were consistent with the Erev values of the initial component of biphasic responses (−87 ± 1.1 mV, n = 26). The time between the initiation and the peak of the inward currents (16 ± 1.2 s, n = 3) for the responses with monophasic decreased gm was significantly faster than the other two types of responses: (35 ± 2.2 s, n = 39 for the biphasic response; 40 ± 3.8 s, n = 12 for the monophasic increased gm response, P = 0.006, Kruskal-Wallis one-way ANOVA on rank). The kinetics of recovery for the monophasic decreased gm responses was relatively slow and varied between neurones. A representative trace along with an I–V plot of this response is shown in Fig. 4A. About 22% of the BK responses were associated with a monophasic increase in gm without discernable decreases in gm (Table 1 and Fig. 3B). The time course and amplitude of these responses were similar to those of the biphasic response. However, their estimated Erev values were significantly less depolarized (24 ± 2.6 mV, n= 12) than the values estimated for the currents associated with the increased gm component of the biphasic BK responses (49 ± 4.3 mV, n= 23). This result could suggest that this increased gm of the monophasic BK response is due to the opening of non-specific cation channels or opening of Cl− channels rather than Na+ and/or Ca2+-specific channels. Alternatively, these differences in Erev values might reflect the presence of two gm during the second component of the biphasic BK response. Thus, depending upon the longevity of the first decreased gm component, the Erev values for the currents associated with the second increased gm component of the biphasic BK responses could be an over-estimation of its true value.

Figure 4. Ion substitution studies for identifying ion(s) responsible for BK-induced inward currents in NGNs.

Figure 4

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A, replacement of extracellular Na+ with NMDG did not abolish BK-induced inward currents in NGNs. In the absence of extracellular Na+, BK (0.1 μm) still evoked inward current related to increased gm, indicating that Na+ is not the main charge carried for BK-induced inward currents in NGNs. B, extracellular Ca2+ was reduced to 0.1 mm and an equimolar concentration of Mg2+ was added. Despite a very the low concentration of extracellular Ca2+ available, BK was able to induce typical inward currents in NGN, suggesting that Ca2+ is unlikely to be a responsible ion for BK-induced inward currents in NGNs. C, to confirm that neither Na+ nor Ca2+ is responsible for BK-induced inward currents, both extracellular Na+ and Ca2+ were replaced by choline and Mg2+ at the same time. The minimum concentration (15.5 mm) of extracellular Na+ was from NaHCO3 (14.3 mm) and NaH3PO4 (1.2 mm). BK still evoked inward currents with increased gm. Horizontal bars without labelling depict the time of BK application.

Ionic mechanisms responsible for BK-induced inward currents

In order to elucidate the ionic mechanisms involved in BK-induced responses, in particular BK responses with increased gm, we altered the extracellular ion composition. Because the Erev values for the currents associated with the increased gm component in the biphasic responses were close to equilibrium potential of Na+ (ENa), ∼70 mV under the present ionic recording conditions, it is possible that Na+ might be an important charge carrier. To test this possibility, we replaced extracellular NaCl with NMDG-Cl or choline-Cl. Surprisingly, we observed large BK-evoked inward currents in the presence of NMDG (0.7 ± 0.30 nA, n = 3) or choline (0.6 ± 0.24 nA, n= 3) despite the presence of a minimum extracellular Na+ concentration (zero Na+ for Hepes-buffered solutions and 15.5 mm Na+ for bicarbonate-buffered solutions) (Fig. 4A). These results indicate that Na+ is unlikely to be a major charge carrier for BK-induced inward currents.

We next lowered extracellular Ca2+ to 0.1 mm, replacing Ca2+ with Mg2+. We did not lower extracellular Ca2+ to zero because complete removal of extracellular Ca2+ can provoke inward current in some NGNs (Undem et al. 2003). In the presence of 0.1 mm Ca2+ (a concentration 20-fold below control levels), BK still induced large inward currents (1.6 ± 0.51 nA, n= 3) with an increase in gm (Fig. 4B). These results suggest that Ca2+ may not be a major charge carrier for the BK responses. It is possible that Na+ might pass through Ca2+ channels when extracellular Ca2+ is reduced or non-specific cation channels might allow either Na+ or Ca2+ to pass through these channels to generate large inward currents. In two NGNs we replaced both extracellular Na+ (15.5 mm) and Ca2+ (0 mm) at the same time with choline and Mg2+. Under these conditions, clear inward currents were observed upon application of BK (0.3 and 1.4 nA, n= 2, Fig. 4C). These results further support the premise that neither Na+ nor Ca2+ is a major charge carrier for BK-induced inward currents in NGNs.

Lastly, we replaced extracellular NaCl with Na-isethionate, an impermeant anion. Under control conditions there were almost equivalent amounts of Cl− intracellularly as there were extracellularly. Thus, with Na-isethionate substitution there should be a shift of ECl from ∼0 mV to ∼66 mV. To diminish the influence of the decreased gm component of the BK response we replaced intracellular K+ with Cs+. In the presence of intracellular Cs+, almost all the BK responses (11/12) were accompanied solely by an increased gm (Table 1 and Fig. 5A). With intracellular Cs+, the Erev value at the peak of the responses was 20 ± 4.7 mV (n= 10), a value similar to the Erev value recorded with a K+-based intracellular solution from monophasic responses associated with increased gm component (24 ± 2.6 mV, n = 12, Table 1). These data further support the conclusion that the decreased gm component of the BK response was related to a K+ current; they are also consistent with our supposition that the initial decreased gm component of the biphasic BK response can contaminate the estimated Erev values for the currents associated with the second increased gm component.

Figure 5. Effect of BK on Cl− currents and their dependence on intracellular Ca2+ rise.

Figure 5

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A, intracellular K+ was replaced with Cs+ in order to isolate the increased gm component from biphasic BK responses in NGNs. In this condition, BK (0.1 μm) induced inward current with increased gm and its Erev was 24 mV. B, extracellular Cl− was replaced with an equimolar concentration of isethionate and Cs+-based internal solution was used. In this ionic condition, BK induced inward current and its Erev value shifted to 55 mV. These results suggested that Cl− is the main charge carrier for BK-induced inward currents in NGNs. C, in order to evaluate the dependence of BK-induced Cl− currents on the intracellular Ca2+ rise, the fast acting Ca2+ chelator, BAPTA (10 mm), was used intracellularly instead of EGTA. In the presence of intracellular BAPTA, BK induced an inward current related to a decreased gm only, suggesting BK-induced Cl− currents were dependent on intracellular Ca2+. The Erev value of this response was −87 mV, which is close to EK. Horizontal bars depict the time of BK application.

When sodium isethionate-containing solutions were used in conjunction with intracellular Cs+, BK induced inward currents (0.2 ± 0.08 nA, n= 4) and, importantly, Erev values shifted significantly to 56 ± 8.8 mV (n= 4, P= 0.002, two-tailed t test, Fig. 5B). Because Erev values estimated by extrapolating I–V plots with linear regression are subject to uncertainties, we also assessed changes in the Erev values using GABA (100 μm), an agonist known to open Cl− channels in neurones (Bormann et al. 1987; Kaila, 1994). Erev of GABA currents in Cs+-based internal solution with control extracellular Cl− was 29 ± 2.9 mV (n = 5), a value similar to Erev for BK responses recorded under comparable ionic conditions (P = 0.336, Mann-Whitney rank sum test). When extracellular NaCl was replaced by Na-isethionate, the Erev of GABA shifted to 64 ± 6.7 mV (n = 5), a value not significantly different from Erev values for BK responses recorded under the same ionic condition (P = 0.528, t test).

Although the Erev values for BK and for GABA were similar to one another, they were significantly different from the calculated ECl of −3 mV. This is probably due to estimation of Erev using extrapolation of the currents evoked by ramp voltage commends from −110 to −50 mV. We used this protocol because (1) depolarizing the membrane potential above −50 mV will activate voltage-gated channels (Na+, Ca2+ and K+), and (2) we could initially substitute impermeant ions for extracellular Na+ and Ca2+ and use ramp voltages positive to −50 mV. However, since Na+ and Ca2+ as well as Cl− were the candidate ions for BK responses, we did not do this experiment until we were able to rule out these two ions. It is known that GABA-generated I–V plots are non-linear with gm increasing with membrane depolarization (Akaike et al. 1985; Weiss et al. 1988; Valeyev et al. 1999). To determine the nature of the disparity between the Erev values for BK and GABA and ECl, we performed additional experiments. In one set of experiments NGNs were held at +70 mV and ramp voltage commands were applied from +70 to −30 mV (1 mV ms−1) during BK and GABA responses recorded with extracellular Hepes-buffered physiological salt solution and a standard patch pipette solution. Under these conditions, the Erev values for BK and GABA were −4 ± 2.2 mV (n = 3) and −1 ± 0.9 mV (n = 5), respectively. In another series of experiments we used a NMDG-based extracellular solution and a Cs+-based pipette solution and applied ramp voltage commands that ranged from −90 to +10 mV (0.5 mV ms−1). The Erev values for BK were −12 and −2 mV (n= 2, Fig. 6) and for muscimol (a GABAA receptor agonist, 100 μm) were 0 and −7 mV (n = 2). These results strongly suggest that at −60 mV, BK can cause an inward current by opening Cl− channels allowing intracellular Cl− to leave the neurones.

Figure 6. Direct measurement of Erev value for a BK response.

Figure 6

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Reversal potential (Erev) value was measured by the intersection of I–V curves recorded in the absence and presence of BK (0.1 μm). To minimize voltage-activated Na+ and K+ currents during membrane depolarization, extracellular Na+ was replaced by NMDG and intracellular K+ by Cs+. The membrane potential was held at −60 mV and ramp voltage commands were applied that ranged from −90 to +10 mV (0.5 mV ms−1). The Erev value for this BK response was −12 mV, close to the calculated ECl (−5 mV).

To evaluate whether the BK-activated Cl− conductance was dependent upon a rise in intracellular Ca2+ following activation of BK receptors, we substituted BAPTA, a fast Ca2+ chelator (Tsien, 1980), for EGTA (Table 1 and Fig. 5C). Under this condition, almost all the responses (6/7) induced by BK were associated with only a decreased gm and showed Erev values of −87 ± 3.6 mV (n = 6). With control ionic condition, it was rare to observe BK responses solely with a decreased gm (3/54, Table 1). These finding suggest that Cl− currents activated by BK were dependent on a rise in intracellular Ca2+.

Pharmacological studies of BK-induced inward currents

We performed several pharmacological manipulations to support the inference that Cl− currents are dependent upon Ca2+ and to identify the nature of the BK receptor(s). NGNs were superfused with niflumic acid (NFA, 100 μm), a Ca2+-activated Cl− channel blocker, for 2 min. When BK was applied, in the presence of NFA, none of the 12 neurones tested showed inward currents associated with an increased gm. Despite severe BK receptor desensitization, we were able to observe, on a few occasions, recovery of BK responses after washout (n = 3). The traces in Fig. 7A show a small BK response with a decreased gm recorded in the presence of NFA. After washout of NFA, the BK-induced inward current was much larger and was associated with an increased gm.

Figure 7. Pharmacological studies of BK responses in NGNs.

Figure 7

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A, a Ca2+-activated Cl− current blocker, niflumic acid (NFA, 100 μm), was applied in order to confirm that BK-induced inward currents were related to increased Cl− conductance. In the presence of NFA, BK (0.1 μm) induced only small inward current, related to decreased gm, that is mediated by inhibition of resting K+ currents. After washout, BK induced an inward current related to an increased gm, further suggesting an involvement of Ca2+-activted Cl− currents in the BK response in NGNs. B, because NFA is known to have anti-inflammatory effects, we used other anti-inflammatory agents, indomethacin (Indo, 5 μm) and ETYA (1 μm), and tried to block cyclooxygenase and lipoxygenase pathways, respectively. In the presence of Indo and ETYA, BK was still able to induce an apparent inward current, suggesting a direct effect of NFA on Cl− channels. C, to rule out a possible involvement of TRPV1 in the BK responses in NGNs, the irreversible TRPV1 antagonist, iodo-resiniferatoxin (iRXT, 0.3 μm) was applied. iRTX failed to block BK-induced inward currents, suggesting TRPV1 is not the ion channel responsible for BK responses in NGNs. D, a selective B2 receptor antagonist, HOE-140 (0.3 μm), was applied before, during and after BK application. HOE-140 completely blocked BK responses with recovery after washout, indicating that BK acted on B2 receptors and evoked inward currents in NGNs. Horizontal bars without labelling depict the time of BK application.

It has been reported that NFA has anti-inflammatory effects by inhibiting cyclooxygenase (COX, Cushman & Cheung, 1976). To clarify whether blockage of BK responses by NFA was secondary to inhibiting COX, we treated NGNs with a known COX inhibitor, indomethacin (5 μm), and with a non-specific lipoxygenase inhibitor, ETYA (1 μm). Pretreatment of NGNs with indomethacin did not attenuate BK-induced responses (1.0 ± 0.53 nA, n = 7). Three of these NGNs were pretreated with ETYA plus indomethacin. All three showed typical inward currents (0.6 ± 0.18 nA) associated with an increased gm following bath-applied BK (Fig. 7B). These results indicate that NFA is likely to be blocking Ca2+-activated Cl− channels. Thus, these data taken in conjunction with the data from ion substitution experiments and the blocking effects of BAPTA strongly suggest that Ca2+-activated Cl− conductance underlies the BK-induced increase in membrane conductance.

Recently, TRPV1 receptors (a vanilloid receptor) have been implicated as a target following BK receptor activation in DRG neurones of neonatal rat (Shin et al. 2002) and in vagus nerve terminals of guinea pig (Carr et al. 2003). To evaluate this possibility, we pretreated NGNs with iodo-resiniferatoxin (iRTX, 0.3 μm), an irreversible TRPV1 antagonist (Wahl et al. 2001), for 2 min. BK-induced inward currents in the presence of iRTX (0.4 ± 0.12 nA, n = 3), suggesting that TRPV1 is unlikely to be involved in BK responses in somata of NGNs (Fig. 7C).

BK can activate two classes of receptors designated B1 and B2 (Couture et al. 2001). Because BK responses rapidly desensitize, studying the effects of BK receptor antagonists can be problematic. Rather than applying BK before the antagonist we pre-incubated NGNs in the presence of HOE-140 (0.3 μm), a B2 receptor antagonist, then subsequently added BK in the presence of the antagonist. Under these conditions, BK never elicited a measurable inward current (7/7). After washout of HOE-140, BK was reapplied. In five NGNs BK evoked inward currents averaging 0.3 ± 0.06 nA (Fig. 7D). Because the BK responses observed upon washout of HOE-140 were accompanied only by an increase in gm, we could not assess whether the decrease gm component of the BK response was also prevented by HOE-140 application. It may that this component desensitizes more rapidly than the increase gm component or that it is activated by B1 receptors. Nonetheless, our data show that B2 receptors in NGNs can activate Ca2+-activated Cl− currents. It is interesting to note that B2 receptors in NGNs can also inhibit Ca2+-activated K+ currents (Weinreich et al. 1995). Whether both BK effects coexist in the same vagal afferent remains to be determined.

Discussion

Our major observation is that BK can excite vagal afferents, NGNs, through two separate ionic mechanisms: inhibition of resting K+ current, and activation of Ca2+-dependent Cl− current. In most NGNs studied, BK evoked an inward current that was associated with two distinct membrane conductances (gm). During the initial few seconds of BK application gm was reduced, and then it increased for tens of seconds. The early component had an estimated reversal potential (Erev) near EK and was abolished when intracellular K+ was replaced by Cs+, indicating that the initial decrease in gm was likely due to the closing of K+ channels. The second and more robust component of BK-evoked inward currents is likely to be mediated by a Ca2+-activated Cl− conductance for the following reasons: (1) it was associated with an increased gm, (2) the estimated Erev shifted in a positive direction following substitution of extracellular NaCl with Na-isethionate, an impermeant anion, (3) the inward current was blocked by intracellular BAPTA, and (4) the second component was inhibited by niflumic acid, a Ca2+-activated Cl− channel blocker. Collectively, these observations support the contention that BK produces an inward current in NGNs through cation and anion channels.

Multiple mechanisms have been proposed for BK's sensitizing and excitatory effects on primary afferent neurones. BK can sensitize sensory neurones by inhibiting a slow afterhyperpolarization (AHPslow) by blocking Ca2+-activated K+ currents, an effect mediated by the production of prostacyclin (Weinreich, 1986; Weinreich et al. 1995). By inhibiting the AHPslow BK can reduce spike accommodation and increase repetitive action potential discharge (Weinreich & Wonderlin, 1987). BK may directly excite primary afferents by opening Na+ channels in a protein kinase C-dependent manner via an inositol phospholipid hydrolysis pathway (Burgess et al. 1989). BK may also activate lipoxygenases producing lipid metabolites that stimulate TRPV1 receptors (a vanilloid receptor) leading to the opening of non-specific cation channels (Shin et al. 2002). The current work reveals an additional mechanism by which BK may increase excitability in primary sensory neurones, namely, modulating anionic (Cl−) as well as cationic (K+) conductances. Though studies in non-neuronal tissues (Kose et al. 2000; England et al. 2001) have documented that BK can trigger Ca2+-activated Cl− conductances, the BK-evoked Cl− conductances recorded in vagal afferent neurones may be the first demonstration of an inflammatory mediator exciting a primary afferent neurone via an anion channel.

Ca2+-activated Cl− channels are expressed in a number of peripheral and central neurones, including visceral and somatic primary afferent neurones (reviewed by Frings et al. 2000). Despite their presence the physiological roles for these channels remain largely unresolved, except for olfactory sensory neurones where they mediate odourant transduction (Schild & Restrepo, 1998). In somatic and visceral primary afferents, BK-evoked Ca2+-activated Cl− currents may participate in at least three functions: (1) nerve injury (axotomy), (2) neurite outgrowth, and (3) amplification of localized signals.

Ca2+-activated Cl− currents are up-regulated in injured (axotomized) sensory neurones, particularly in large diameter (presumably non-nociceptive) neurones (Lancaster et al. 2002; Andréet al. 2003). Assuming that BK receptors are present and functional in axotomized neurones, BK produced during tissue injury could excite non-nociceptive (as well as nociceptive) afferents by opening Ca2+-activated Cl− channels up-regulated following nerve injury. This action of BK could underlie abnormal painful sensations in response to previously non-noxious stimuli, a phenomenon designated as allodynia. In this connection it is interesting to note that following axotomy of vagal motor neurones the K+–Cl− cotransporter that moves K+ and Cl− out of the cells (KCC2) is downregulated causing an accumulation of intracellular Cl− and exaggerated excitatory responses to GABA (Nabekura et al. 2002).

Another potential role for BK-triggered Ca2+-activated Cl− currents could involve modulation of cell growth during neuronal regeneration after injury. In developing and regenerating neurones, the growth cone, located at the tip of the neurite, controls nerve growth and axonal guidance (Goodman & Shatz, 1993). IP3-sensitive intracellular Ca2+ stores in the growth cone have been shown to play an important role in signalling during neural regeneration (Takei et al. 1998). It is well documented that BK receptors signal IP3 Ca2+ pools, and thus, BK could serve as a stimulus for neuronal growth. In support of this possibility are the observations that BK can cause neurite extension via IP3-sensitive Ca2+ signalling upon activation of B2 receptors in pheochromocytoma (PC12) cells (Kozlowski et al. 1989; Reber & Schindelholz, 1996; Schindelholz & Reber, 1997).

Due to the presence of a Na+–K+–Cl− cotransporter (NKCC1) mature primary afferent neurones, unlike most adult CNS neurones, have intracellular Cl− concentrations much larger than those predicted by passive distribution of Cl− (Sung et al. 2000). The elevated intracellular Cl− concentration provides the driving force to generate inward currents when Cl− channels are opened. Increasing neuronal excitability through Ca2+-activated Cl− conductances (outward Cl− movement) rather than inward cationic (Na+, Ca2+) conductances might be beneficial for several reasons. First, an effect produced by a small localized influx of Ca2+ or a localized rise in intracellular Ca2+ concentration could be amplified by activating Cl− currents. This might be an effective way of minimizing loss of small signals. Second, when neurones grow through a hypo-osmolar extracellular milieu they can still be effectively depolarized via Cl− currents while depolarizing currents mediated by cations may be compromised. It is noteworthy that Ca2+-activated Cl− channels are widely expressed in cells involved in water and salt transport (for example, renal epithelial cells). Third, unlike cell bodies, other neuronal compartments have small aqueous volume (dendritic spines, nerve terminals) whose intracellular ionic composition may rapidly change with ion fluxes. By depolarizing the membranes of small compartments via Cl− efflux, these compartments would still retain chemical gradients for Na+ and Ca2+. Finally, in order to significantly change intracellular volume, it is necessary to have a flux of both cations and anions; otherwise, electroneutrality would severely limit the number of ions transferred. This raises the possibility that Ca2+-activated Cl− channels help regulate growth cone volume or morphology.

In conclusion, this study investigated the excitatory actions of BK on vagal primary afferent neurones. In the same primary afferent, BK blocked a resting K+ conductance and promoted a Ca2+-activated Cl− conductance. Our findings reveal a previously undescribed role for Ca2+-activated Cl− channels expressed in visceral sensory neurones; namely, their participation in an excitatory signalling pathway for inflammatory mediators.

Acknowledgments

We would like to thank Dr Michael Gold for his valuable input to this work and for his critique of an earlier version of this manuscript. This work was supported by NIH grants NS22069 (D.W.).

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