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Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Baltimore, MD, USA
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Abstract |
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(Received 11 April 2004;
accepted after revision 24 May 2004;
first published online 28 May 2004)
Corresponding author D. Weinreich: University of Maryland School of Medicine, Department of Pharmacology and Experimental Therapeutics, Room 4-002, Bressler Research Building, 655 West Baltimore Street, Baltimore, MD 21201-1559, USA. Email: dweinrei{at}umaryland.edu
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Introduction |
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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.
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Methods |
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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 M
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(ß-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 M, Rm > 100 M, 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.
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Results |
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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 40x; 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.
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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.
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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.
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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.
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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.
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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.
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Discussion |
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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.
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