Inhibition of KATP channels in the rat tail artery by neurally released noradrenaline acting on postjunctional α2-adrenoceptors
- 1Prince of Wales Medical Research Institute, University of New South Wales, Barker Street, Randwick, NSW 2031, Australia
- Corresponding author J. Brock: Prince of Wales Medical Research Institute, Barker St, Randwick, NSW 2031, Australia. Email: j.brock{at}unsw.edu.au
Abstract
In rat tail artery, activation of postjunctional α2-adrenoceptors by noradrenaline (NA) released from sympathetic axons produces a slow depolarization (NAD) of the smooth muscle through a decrease in K+ conductance. In this study we used intracellular recording to investigate whether the K+ channel involved is the ATP-sensitive K+ (KATP) channel. Changes in membrane resistance were monitored by measuring the time constant of decay of excitatory junction potentials. The KATP channel blockers, glibenclamide (10 μm) and PNU 37883A (5 μm), depolarized the smooth muscle and increased membrane resistance. Conversely, the KATP channel openers, pinacidil (0.1 and 0.5 μm) and levcromakalim (0.1 μm), hyperpolarized the smooth muscle and decreased membrane resistance. Activation of KATP channels with calcitonin gene-related peptide (CGRP; 10 nm) also hyperpolarized the smooth muscle and decreased membrane resistance. The NAD was abolished by both glibenclamide and PNU 37883A but was potentiated by CGRP. However, unlike CGRP, the directly acting KATP channel openers, pinacidil and levcromakalim, inhibited the NAD. The effects of other K+ channel blockers were also determined. A high concentration of Ba2+(1 mm), which would be expected to block KATP channels, abolished the NAD, whereas teteraethylammonium (1 mm) and 4-aminopyridine (1 mm) increased its amplitude. Apamin (0.5 μm) and a lower concentration of Ba2+ (0.1 mm) did not affect the NAD. These findings indicate that activation of α2-adrenoceptors by neurally released NA depolarizes the membrane of vascular smooth muscle by inhibiting KATP channels open in the resting membrane.
Activation of α1-adrenoceptors in both vascular and non-vascular smooth muscles produces depolarization through activation of Ca2+-activated Cl− channels (Large & Wang, 1996) and transient receptor potential (TRP)-like cation channels (Large, 2002; Albert & Large, 2006). The ionic mechanisms that underlie α2-adrenoceptor-mediated depolarization of smooth muscle have not been studied directly, but it has been proposed that activation of these receptors reduces the activity of ATP-sensitive K+ (KATP) channels in skeletal muscle arterioles (Tateishi & Faber, 1995). This suggestion was based on the observation that constriction of these arterioles in response to the KATP channel blocker glibenclamide occluded that in response to the α2-adrenoceptor agonist UK 14 304, and vice versa. Contractions in response to the α1-adrenoceptor agonist phenylephrine were unaffected by glibenclamide. The actions of nerve-released noradrenaline (NA) were not studied in these arterioles.
In rat tail artery, nerve stimulation evokes both a fast purinergic excitatory junction potential (EJP) and a slow noradrenergic depolarization (NAD) that is due primarily to activation of postjunctional α2-adrenoceptors (Itoh et al. 1983; Cassell et al. 1988). In this vessel, EJPs evoked at the peak of the NAD are little affected in amplitude but their time constant of decay (τEJP) is markedly prolonged (Cassell et al. 1988). The depolarization produced by the α2-adrenoceptor agonist, clonidine, is also associated with an increase in τEJP but, in addition, this agent reduces EJP amplitude through an action at prejunctional α2-adrenoceptors (Brock & Tan, 2004). In rat tail artery, the τEJP is similar to the membrane time constant and its prolongation most probably indicates an increase in membrane resistance (Cassell et al. 1988); the membrane time constant is the product of membrane resistance and capacitance. Therefore the increase in τEJP during both the NAD and the application of clonidine indicates that α2-adrenoceptor-mediated depolarization results from a decrease in membrane K+ conductance. An α2-adrenoceptor-mediated reduction in K+ conductance has also been suggested to underlie the slow component of neurally evoked depolarization in capsular smooth muscle of rat and guinea-pig spleen (Jobling, 1994).
In the present study we have used electrophysiological techniques to investigate whether the decreased membrane conductance produced by neurally released NA is due to inhibition of KATP channels in the rat tail artery. We found that the KATP channel blockers, glibenclamide and PNU 37883A, reduced the resting membrane potential (RMP) and abolished the NAD. These finding indicate (1) that KATP channels contribute to setting the RMP and (2) that activation of α2-adrenoceptors by neurally released NA inhibits the basal activity of KATP channels, resulting in membrane depolarization.
Methods
All experimental procedures conformed to the National Health and Medical Research Council of Australia guidelines and were approved by the University of New South Wales Animal Care and Ethics Committee.
Female inbred Wistar rats (200–250 g) were anaesthetized (100 mg kg−1 pentobarbitone, i.p.) and killed by exsanguination. The main ventral caudal artery was dissected from 10 to 40 mm distal to the base of the tail. Approximately 15 mm segments of artery were pinned to the Sylgard (Dow Corning)-coated base of a 1 ml recording chamber. The chamber was perfused continuously at 3–5 ml min−1 with physiological saline containing (mm): Na+ 150.6, K+ 4.7, Ca2+ 2, Mg2+ 1.2, Cl− 144.1, H2PO4− 1.3, HCO3− 16.3 and glucose 7.8. This solution was gassed with 95% O2–5% CO2 and warmed to 35–36°C (pH 7.25). In all experiments, the physiological saline contained the α1-adrenoceptor antagonist, prazosin (0.1 μm), to inhibit neurally evoked contraction due to NA release. Prazosin at this concentration does not change the RMP (control, − 61 ± 2 mV; prazosin, −61 ± 2 mV, n = 5, P = 0.97, paired t test). The proximal end of the artery was drawn into a suction stimulating electrode and the perivascular nerves excited by electrical field stimulation (1 ms pulse width, 20 V). As increasing stimulus voltage did not increase the size of the signals recorded, these stimulus parameters were assumed to be supramaximal.
Electrophysiological recording
Intracellular recordings were made using glass microelectrodes (120–200 MΩ) filled with 0.5 m KCl and connected to an Axoclamp bridge amplifier (Axon Instruments, Union City, CA, USA). To avoid EJPs with an early fast component recorded in cells close to the sites of neuromuscular junctions, recordings were made from cells located deeper in the media in which EJPs decayed mono-exponentially, reflecting the electrical behaviour of the smooth muscle syncytium (see Cassell et al. 1988). Impalements were only accepted if the following criteria were satisfied: (1) the cell penetration was abrupt; (2) the membrane potential increased to a value more negative than the initial potential; (3) the membrane potential was stable; and (4) electrical stimulation evoked EJPs. RMP was determined upon withdrawal of the microelectrode.
Experimental protocols
In all electrophysiology experiments, recordings were first made from three or four cells under control conditions. During each recording, arteries were stimulated at 1 min intervals with five stimuli at 1 Hz. The drug was then added to the superfusing solution and allowed to equilibrate with the tissue for at least 10 min before recordings from another three or four cells were obtained. In the experiments with pinacidil, the effects of 0.1 μm followed by 0.5 μm were studied, with recordings from three or four cells at each concentration.
Electrochemical recording
The release of endogenous NA was monitored by continuous amperometry using a technique similar to that described by Dunn et al. (1999). This technique measures the stimulus-evoked increase in NA at the adventitial surface of the artery as an oxidation current, the amplitude of which is linearly related to NA concentration. Briefly, Nafion-coated carbon fibre electrodes (7 μm diameter and 150–200 μm in length) were mounted at about 30 deg to the horizontal and placed gently against the surface of the artery so that an approximately 100 μm length of carbon fibre was in contact with the tissue. The electrode was connected to an AMU130 Nano-amperometer (Pacific Radiometer, Blackburn, Victoria, Australia) and a potential difference of +0.3 V was applied between the recording electrode and an Ag–AgCl pellet placed in the recording chamber medium. The current required to maintain this voltage was monitored. After placement of the carbon fibre electrode, the preparation was left for 20–30 min before starting the experiment.
During the experiment, tissues were stimulated at 2 min intervals with 10 stimuli at 10 Hz, with pinacidil or glibenclamide added following the tenth train of stimuli and left in contact with the tissue for a further 10 trains of stimuli. Three responses recorded just before (T1) and 16–20 min after the addition of the drug (T2) were averaged before measurements were made. In control experiments in which no drugs were added, the amplitude of the oxidation current declined slightly between T1 and T2. To account for this time-dependent change in the amplitude of the oxidation currents, T2/T1 ratios for the drug-treated tissues were compared with those obtained in the control tissues.
Data analysis
All data were digitized (sampling frequencies of 0.2 kHz) and collected with a PowerLab recording system and the program Scope (ADInstruments, Castle Hill, NSW, Australia). Subsequent analysis was made using Igor Pro (Wavemetrics, Lake Oswego, OR, USA). The peak amplitude and time constant of decay of the first EJP evoked during the trains of stimuli at 1 Hz was determined. The τEJP was estimated by fitting a mono-exponential function to the decay phase of the EJP. The peak amplitude of the slow depolarization was also measured. For each artery, electrophysiological data obtained under control conditions and in the presence of the drug were averaged before statistical comparisons were made using Student's paired t tests. For the experiments investigating the effects of 0.1 and 0.5 μm pinacidil, the data were first compared using a repeated measures ANOVA and the P values for the pairwise comparisons were corrected using the Dunn–Sidák method. For the electrochemistry experiments, one-way ANOVA was used to compare the T1/T2 ratios for the control tissues with those obtained in tissues treated with pinacidil or glibenclamide.
Unless otherwise indicated, data are presented as mean ± s.e.m. For all statistical tests, P < 0.05 was taken as a significant difference. In all cases, n refers to the number of preparations studied.
Drugs
Glibenclamide, levcromakalim, prazosin HCl, PNU 37883A, 4-aminopyridine and tetraethylammonium were supplied by Sigma Chemical Company (Castle Hill, NSW, Australia). Pinacidil was supplied by Tocris Cookson Ltd (Bristol, UK), rat calcitonin gene-related peptide1–37 (CGRP) by Auspep Pty Ltd (Parkville Victoria, Australia) and apamin by Alomone Labs (Jerusalem, Israel). Prazosin was prepared as a 1 mm stock solution in 10% (v/v) dimethylsulphoxide (DMSO) in water and glibenclamide, levcromakalim and pinacidil were prepared as 10 mm stock solutions in DMSO. In the experiments, 0.1% (v/v) DMSO was the highest concentration used and this was without effects on RMP or the electrically evoked responses (Table 1). All the other drugs were prepared as stock solutions in water.
Results
General observations
Under control conditions (n = 54 tissues), each stimulus during trains of five stimuli at 1 Hz evoked an EJP lasting about 1 s and successive EJPs increased in amplitude during the train (Fig. 1). The amplitude of the first EJP in the train was 10.0 ± 2.7 mV (mean ± s.d.) and its time constant of decay was 248 ± 45 ms. Stimulation also produced an NAD (1.3 ± 0.6 mV) that peaked 15–20 s after the first stimulus in the train and lasted about 1 min (Fig. 1). The RMP was −66 ± 3 mV.
Effects of the KATP blockers glibenclamide and PNU 37883A
Both glibenclamide (10 μm, n = 7; Fig. 1) and PNU 37883A (5 μm, n = 6) produced a small depolarization of the membrane (Table 1). In the presence of these agents, there was also a small reduction in EJP amplitude and an increase in τEJP (Table 1 and Fig. 1). In addition, both glibenclamide (Fig. 1) and PNU 37883A abolished the NAD (Table 1).
Effects of pinacidil and levcromakalim
The KATP channel openers, pinacidil (0.1 and 0.5 μm, n = 8; Fig. 2) and levcromakalim (0.1 μm, n = 6), produced a hyperpolarization of the membrane (Table 1) and a decrease in τEJP (Table 1). In addition, pinacidil and levcromakalim produced a small decrease in EJP amplitude and more markedly reduced the amplitude of the NAD (Table 1).
CGRP augmented the NAD
As with pinacidil and levcromakalim, CGRP (10 nm, n = 6; Fig. 3) produced both a hyperpolarization of the membrane and a decrease in τEJP (Table 1), but did not significantly change EJP amplitude (Table 1, P = 0.33). However, unlike pinacidil and levcromakalim, the amplitude of the NAD was increased by CGRP (Table 1 and Fig. 3).
Glibenclamide blocked the effects of pinacidil and CGRP
Neither pinacidil (0.5 μm, n = 8) nor CGRP (10 nm, n = 6) changed the membrane potential in the presence of glibenclamide (pinacidil, changed by 1.2 ± 0.6 mV, P = 0.23; CGRP, changed by 0.2 ± 0.4 mV, P = 0.93). Furthermore, when glibenclamide was present, τEJP was not changed by the addition of pinacidil (glibenclamide, 302 ± 12 ms; glibenclamide + pinacidil, 289 ± 12 ms, P = 0.15) or CGRP (glibenclamide, 293 ± 13 ms; glibenclamide + CGRP, 292 ± 11 ms, P = 0.65). These findings confirm that both the hyperpolarization and the decrease in τEJP produced by these agents were due to activation of glibenclamide-sensitive K+ channels (Standen et al. 1989; Quayle et al. 1994).
Neither pinacidil nor glibenclamide changed NA-induced oxidation currents
In control experiments, the amplitude of the NA-induced oxidation current evoked by 10 stimuli at 10 Hz declined slightly between T1 and T2 (T2/T1 ratio, 0.86 ± 0.07, n = 6; (Fig. 4). The T2/T1 ratios in the experiments with pinacidil (0.5 μm; 0.83 ± 0.02, n = 6) or glibenclamide (10 μm; 0.87 ± 0.04, n = 6) were similar to those obtained in the control experiments (P = 0.85, one-way ANOVA; Fig. 4), indicating that these agents did not detectably change NA release.
Effects of other K+ channel blockers on neurally evoked electrical activity
The effects of the K+ channel blockers Ba2+, tetraethylammonium (TEA), 4-aminopyridine (4-AP) and apamin, which block a range of K+ channels present in the smooth muscle and endothelium of rat tail artery (Bolzon et al. 1993; Rembold & Chen, 1998; Sandow et al. 2003), were also assessed (Table 2). Ba2+ (0.1 mm, n = 6) reduced both RMP and EJP amplitude and increased τEJP but did not significantly change the amplitude of the NAD (Table 2). At 1 mm (n = 4), Ba2+ almost abolished the NAD and this change was associated with a larger decrease in RMP and a larger increase in τEJP (Table 2).
Both TEA (1 mm, n = 5) and 4-AP (1 mm, n = 6) reduced RMP and increased EJP and NAD amplitude (Table 2). Neither TEA nor 4-AP significantly changed τEJP. In the presence of either TEA (n = 4) or 4-AP (n = 4), we confirmed that the addition of glibenclamide (10 μm) reduced RMP (TEA, −60 ± 2 mV; TEA + glibenclamide, −57 ± 2 mV, P < 0.032; 4-AP, −61 ± 1 mV; 4-AP + glibenclamide, −58 ± 1 mV, P = 0.017), increased τEJP (TEA, 251 ± 16 ms; TEA + glibenclamide, 278 ± 21 ms, P = 0.017; 4-AP, 255 ± 10 ms; 4-AP + glibenclamide, 304 ± 18 ms, P < 0.029) and abolished the NAD. Apamin (0.5 μm, n = 5) had no effect on any of the measured parameters (Table 2).
Discussion
KATP channels are expressed by the vascular smooth muscle of rat tail artery (Cao et al. 2002; Sun et al. 2004) and, as both glibenclamide and PNU 37883A produced depolarization of this tissue, these channels are likely to contribute to setting the RMP. Consistent with this suggestion, glibenclamide and PNU 37883A also increased τEJP, indicating that the depolarization produced by these agents is associated with an increase in membrane resistance (see Introduction). Conversely, increasing the activity of KATP channels with pinacidil, levcromakalim or CGRP produced both a hyperpolarization of the smooth muscle and a decrease in the τEJP that is indicative of a decrease in membrane resistance. In tail artery, the α2-adrenoceptor-mediated NAD is known to result from a decrease in K+ conductance (Cassell et al. 1988). Therefore the finding that the NAD was abolished by glibenclamide and PNU 37883A, and potentiated by increasing KATP activity with CGRP, makes it likely that activation of α2-adrenoceptors produces depolarization of the smooth muscle by inhibiting the activity of KATP channels.
At normal intracellular ATP levels, it has been suggested that the basal activity of KATP channels results from steady-state phosphorylation by cAMP-dependent protein kinase (PKA) and that inhibition of this enzyme by reducing cAMP levels reduces the activity of these channels (Hayabuchi et al. 2001). While the postjunctional subtype of the α2-adrenoceptor activated by neurally released NA in the rat tail artery is not known, all subtypes of α2-adrenoceptors are coupled through G-proteins of the Gi/Go family to the inhibition of adenylyl cyclase (Guimaraes & Moura, 2001). Therefore, the simplest explanation for the effects of activating α2-adrenoceptors is inhibition of adenylyl cyclase and a resultant decrease in the PKA activity. CGRP is reported to cause KATP channel activation through stimulation of adenylyl cyclase and the resultant increase in activity of PKA (Quayle et al. 1994). In this stimulated state where PKA activity is increased, activation of α2-adrenoceptors might produce a larger inhibition of PKA activity and this would explain why the amplitude of the NAD increased. However, in preliminary experiments, cell-permeant inhibitors of PKA (H-89, 8-bromo-Rp-cAMP and KT5720) had no inhibitory effect on either the NAD or the CGRP-induced hyperpolarization (authors' unpublished observations). Therefore the mechanisms that couple activation of α2-adrenoceptors to modulation of KATP channels require further investigation.
Whereas activation of the KATP channels with CGRP increased the amplitude of the NAD, pinacidil and levcromakalim reduced the amplitude of this signal. This difference cannot simply be explained by differences in the level of KATP channel activation, as both CGRP and the lower concentration of pinacidil produced similar changes in RMP and in τEJP (Table 1). Both pinacidil and levcromakalim act directly to activate KATP channels by binding to the sulphonylurea receptor subunits of the KATP channel (Ashcroft & Gribble, 2000). It is known that reducing basal levels of PKA activity can inhibit the pinacidil-induced KATP current in isolated smooth muscle cells (e.g. Hayabuchi et al. 2001). Therefore, the reduction in amplitude of the NAD by pinacidil and levcromakalim may indicate that α2-adrenoceptor modulation of KATP channels is mediated through a mechanism that does not involve inhibition of adenylyl cyclase. Alternatively, the reduction in cAMP levels produced by neuronally released NA may not be sufficient to inhibit the K+ current induced by pinacidil and levcromakalim. It is noteworthy that although α2-adrenceptor-mediated contractions of skeletal muscle arterioles were occluded by glibenclamide, they were more sensitive to the inhibitory actions of KATP activator cromakalim than α1-adrenceptor-mediated contractions (Tateishi & Faber, 1995). This finding indicates that α2-adrenoceptor activation is ineffective in reducing the cromakalim-induced increase in KATP channel activity in skeletal muscle arterioles.
The change in membrane resistance produced by activating or blocking KATP channels would be expected to change the amplitude of EJPs generated by constant junctional currents. However, as the duration of the junctional current is much briefer than that of the EJP (Åstrand et al. 1988), the change in membrane resistance would only slightly affect the impedance seen by the excitatory junction current, as much of the current flow will be capacitive (see Edwards et al. 1976). In Fig. 5 the effects of changing membrane resistance on EJP amplitude have been modelled and the relatively small decrease in EJP amplitude produced by pinacidil and levcromakalim can probably be explained by the decrease in membrane resistance produced by these agents (cf. Fig. 3). This model does not account for the effects of changing RMP on the junctional current. However, with EJPs of approximately 10 mV amplitude and changes in RMP that range between +5 and −10 mV (from −65 mV), the size of the underlying current (reversal potential, ∼0 mV; Finkel et al. 1984) would be little affected (∼2% change; McLachlan & Martin, 1981).
The decrease in EJP amplitude produced by glibenclamide and PNU 37883A cannot be explained by the increase in smooth muscle membrane resistance produced by these agents (see Fig. 5). Therefore it is likely that both glibenclamide and PNU 37883A reduced the junctional current. It is unlikely that this inhibitory effect is due to a reduction in neurotransmitter release, because: (1) blockade of nerve terminal KATP channels would be expected to increase neurotransmitter release (Oe et al. 1999; Burgdorf et al. 2004); and (2) neither pinacidil nor glibenclamide detectably changed NA release. However, we cannot exclude the possibility that glibenclamide and PNU 37883A act selectively to reduce ATP release. Alternatively, these agents may act postjunctionally to reduce the P2X-purinoceptor current.
Decreasing the resting membrane K+ conductance with 0.1 mm Ba2+, which blocks inward rectifier K+ channels, produced a larger reduction in RMP than that produced by either glibenclamide or PNU 37883A but did not change the NAD (see Tables 1 and 2). Therefore the inhibitory effects of KATP channel blockers on the NAD cannot be attributed to depolarization-induced closure of inward rectifier K+ channels. Increasing the concentration of Ba2+ to 1 mm, which would be expected to substantially block KATP channels (IC50, ∼0.5 mm; Teramoto et al. 2006), more markedly reduced the RMP and membrane conductance and in addition almost abolished the NAD. Blockade of either BK Ca2+-activated K+ channels or voltage-dependent K+ channels with TEA and 4-AP, respectively, increased the amplitude of both the EJP and the NAD. These effects of TEA and 4-AP are readily explained by their facilitatory action on NA and ATP release from the sympathetic nerve terminals (Msghina et al. 1998). TEA and 4-AP also reduced the RMP but, as this effect was not associated with an increase in τEJP, it does not appear to result from a reduction in smooth muscle K+ conductance. A possible explanation is that, under resting conditions, these agents have a selective action on endothelial K+ channels and the resulting depolarization spreads electrotonically to the smooth muscle (Sandow et al. 2003; Coleman et al. 2004). While both TEA and 4-AP are reported to block KATP channels, concentrations > 1 mm are usually required for this action in intact preparations (e.g. see Wilson et al. 1988). Apamin, a blocker of SK Ca2+-activated K+ channels, had no effects. Together these findings further support our conclusion that inhibitory effects of glibenclamide and PNU 37883A on the NAD are due inhibition of KATP channels.
The small depolarization produced by blocking KATP channels would not be expected to greatly increase the opening probability of Ca2+ channels and it is therefore unlikely to produce contraction of the smooth muscle. Accordingly, we have not observed contraction in response to glibenclamide in ring segments of rat tail artery mounted isometrically (authors' unpublished observations). However, the increase in membrane resistance and the associated prolongation of the membrane time constant may enhance the actions of other vasoconstrictor agents. For example, increase in the membrane time constant will cause the purinergic EJPs to summate more readily during repetitive activity, increasing both the magnitude of the membrane depolarization and the likelihood of opening voltage-gated Ca2+ channels (e.g. see Cassell et al. 1988). The increase in membrane resistance may also amplify depolarizations produced by other vasoconstrictor agents. This latter effect may explain the facilitatory actions of α2-adrenoceptor activation on contractions in response to a range of vasconstrictor agents (α1-adrenoceptor agonists, vasopressin and ATP) in the rat tail artery (Xiao & Rand, 1990). It is important to note that this synergistic action of α2-adrenoceptor activation is dependent on opening of voltage-dependent Ca2+ channels, presumably because of an augmentation of membrane depolarization (Xiao & Rand, 1989, 1990).
In addition to enhancing the effects of vasconstrictor agents, the inhibition of KATP channels produced by α2-adrenoceptor activation would be expected to oppose the actions of vasodilator agents such as CGRP that increase the KATP channel activity. However, it is worth noting that CGRP is unlikely to be a physiological regulator of smooth muscle tone in the tail artery, as the perivascular nerve plexus contains very few peptidergic afferent axons and this artery lacks vasodilator responses to afferent nerve activation (Li & Duckles, 1993).
In conclusion, activation of α2-adrenoceptors by neurally released NA produces membrane depolarization in the rat tail artery by reducing the activity of KATP channels. Although this action is unlikely to modify the activity of vascular smooth muscle directly, inhibition of KATP channel activity may act indirectly both to potentiate the actions of vasoconstrictor agents that depolarize the vascular smooth muscle and to oppose the effects of vasodilator agents that increase KATP channel activity.
Acknowledgements
This work was supported by the National Health and Medical Research Council of Australia (Project Grant no. 350903, Fellowship Grant no. 350904). We thank Elspeth McLachlan for her comments on the manuscript.
Footnotes
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(Resubmitted 30 January 2007; accepted after revision 19 March 2007; first published online 22 March 2007)
- 2007 The Authors. Journal compilation © 2007 The Physiological Society
References
Figure 1. Glibenclamide abolished the NAD and slowed the rate of decay of EJPs Traces of electrical responses to five stimuli at 1 Hz before (control) and during application of glibenclamide (10 μm). The inset shows the EJPs evoked by the first stimulus in the train overlaid on an expanded time scale. In this tissue, the resting membrane potential before and during application of glibenclamide was –67 mV and –62 mV, respectively.
Figure 2. Pinacidil accelerated the rate decay of EJPs but reduced the amplitude of the NAD Traces of electrical responses to five stimuli at 1 Hz before (control) and during the application of 0.1 and 0.5 μm pinacidil. The inset shows the EJPs evoked by the first stimulus in the train overlaid on an expanded time scale. In this tissue, the resting membrane potential before and during application of 0.1 and 0.5 μm pinacidil was −65, −74 and −76, respectively.
Figure 3. Calcitonin gene-related peptide (CGRP) increased the amplitude of the NAD and accelerated the rate of decay of EJPs A, overlaid traces of electrical responses to five stimuli at 1 Hz before (control) and during the application of CGRP (10 nm). B, the EJPs evoked by the first stimulus in the train overlaid on an expanded time scale. In this tissue, the resting membrane potential before and during application of CGRP was –61 mV and –66 mV, respectively.
Figure 4. Neither glibenclamide nor pinacidil changed noradrenaline-induced oxidation currents evoked by electrical stimulation Overlaid traces of NA-induced oxidation currents evoked by 10 stimuli at 10 Hz at T1 and T2 in control experiments and in experiments with glibenclamide (10 μm) or pinacidil (0.5 μm) added at T2.
Figure 5. Changing membrane resistance has relatively little effect on amplitude of modelled EJPs EJPs were mathematically calculated on the assumption of Edwards et al. (1976) that an excitatory synapse can be described as a constant current generator and the transmembrane current has both a resistive and a capacitive component. Assuming that the specific membrane capacitance is 1 μF cm−2, the cell membrane was initially modelled so that its time constant was 250 ms by setting the specific membrane resistance (Rm) to 250 kΩ cm2. For all calculations, the resting membrane potential was set at −65 mV and the rise time and time constant of decay of the junctional current was set to 10 and 25 ms, respectively (see Åstrand et al. 1988). The current amplitude was scaled to give an EJP of approximately 10 mV under the initial conditions. A, shows the changes in amplitude and time course of modelled EJPs produced by increasing and decreasing membrane resistance by 40% from its initial value. B, shows graphically the relative changes in EJP amplitude for changes in membrane resistance that range between 0.5 and 1.5 of the initial value.
Table 1. Effects of KATP channel blockers and activators on resting membrane potential and neurally evoked electrical activity
Table 2. Effects of Ba2+, tetraethylammonium, 4-aminopyridine and apamin on resting membrane potential and neurally evoked electrical activity
