Muscarinic M 1 receptors activate phosphoinositide turnover and Ca 2+ mobilisation in rat sympathetic neurones, but this signalling pathway does not mediate M‐current inhibition
1999; Wiley; Volume: 520; Issue: 1 Linguagem: Inglês
10.1111/j.1469-7793.1999.00101.x
ISSN1469-7793
AutoresE. Del Río, Jorge A. Bevilacqua, Stephen J. Marsh, Pamela A. Halley, Malcolm P. Caulfield,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoThe Journal of PhysiologyVolume 520, Issue 1 p. 101-111 Free Access Muscarinic M1 receptors activate phosphoinositide turnover and Ca2+ mobilisation in rat sympathetic neurones, but this signalling pathway does not mediate M-current inhibition Elena del Río, Elena del Río Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UKSearch for more papers by this authorJorge A. Bevilacqua, Jorge A. Bevilacqua ICBM-Facultad de Medicina, Universidad de Chile, Independencia 1027, cc 70079, Santiago, ChileSearch for more papers by this authorStephen J. Marsh, Stephen J. Marsh Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UKSearch for more papers by this authorPamela Halley, Pamela Halley Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UKSearch for more papers by this authorMalcolm P. Caulfield, Corresponding Author Malcolm P. Caulfield Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UKCorresponding author E. del Río: Wolfson Institute for Biomedical Research, University College London, 1 Wakefield Street, London WC1N 1PJ, UK. Email: rmgzed[email protected]Search for more papers by this author Elena del Río, Elena del Río Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UKSearch for more papers by this authorJorge A. Bevilacqua, Jorge A. Bevilacqua ICBM-Facultad de Medicina, Universidad de Chile, Independencia 1027, cc 70079, Santiago, ChileSearch for more papers by this authorStephen J. Marsh, Stephen J. Marsh Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UKSearch for more papers by this authorPamela Halley, Pamela Halley Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UKSearch for more papers by this authorMalcolm P. Caulfield, Corresponding Author Malcolm P. Caulfield Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UKCorresponding author E. del Río: Wolfson Institute for Biomedical Research, University College London, 1 Wakefield Street, London WC1N 1PJ, UK. Email: [email protected]Search for more papers by this author First published: 07 September 2004 https://doi.org/10.1111/j.1469-7793.1999.00101.xCitations: 38 Authors' present addresses: P. Halley: Biochemistry Department, University of Dundee, Dundee, UK. M. P. Caulfield: Faculty of Law, University of Western Australia, Perth, Western Australia, 6907 Australia. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract 1 The relationship between muscarinic receptor activation, phosphoinositide turnover, calcium mobilisation and M-current inhibition has been studied in rat superior cervical ganglion (SCG) neurones in primary culture. 2 Phosphoinositide-specific phospholipase C (PLC) stimulation was measured by the accumulation of [3H]-cytidine monophosphate phosphatidate (CMP-PA) after incubation with [3H]-cytidine in the presence of Li+. The muscarinic agonist oxotremorine methiodide (oxo-M) stimulated PLC in a dose-dependent manner with an EC50 of approximately 3.5 μm. 3 The concentration-response curve for oxo-M was shifted to the right by a factor of about 10 by pirenzepine (100 nm), suggesting a pKB (—log of the apparent dissociation constant) of 7.9 ± 0.4, while himbacine (1 μm) shifted the curve by a factor of about 13 (pKB∼7.1 ± 0.6). This indicates involvement of the M1 muscarinic receptor in this response. 4 The accumulation of CMP-PA was localised by in situ autoradiography to SCG principal neurones, with no detectable signal in glial cells present in the primary cultures. 5 The ability of oxo-M to release Ca2+ from inositol(1,4,5)trisphosphate (InsP3)-sensitive stores was determined by fura-2 microfluorimetry of SCG neurones voltage clamped in perforated patch mode. Oxo-M failed to evoke intracellular Ca2+ (Ca2+i) mobilisation in SCG neurones voltage clamped at −60 mV, but produced a significant Ca2+i rise (67 ± 15 nm, n= 9) in cells voltage clamped at −25 mV. 6 Thapsigargin (0.5–1 μm) caused a 70% inhibition of the oxo-M-induced Ca2+i increase, indicating its intracellular origin, while oxo-M-induced inhibition of M-current in the same cells was unaffected by thapsigargin. 7 Our results do not support the involvement of InsP3-sensitive calcium mobilisation in M-current inhibition. In rat sympathetic neurones, a major electrophysiological consequence of activation of M1 muscarinic acetylcholine receptors is the inhibition of a non-inactivating time- and voltage-dependent K+ current, the M-current (IK(M)), with a resultant slow depolarisation and a dramatic increase in neuronal excitability (Brown & Selyanko, 1985; for reviews, see Brown, 1988; Marrion, 1997). Muscarinic receptor agonists also activate phsopholipase C (PLC) in intact SCG (Bone et al. 1984; Horwitz et al. 1985; Ramcharan & Matthews, 1996). These two effects are likely to involve muscarinic receptor interaction with the same G-protein, Gαq (Smrcka et al. 1991; Caulfield et al. 1994; Haley, Abogadie et al. 1998; reviewed by Caulfield, 1993). It has been shown that muscarinic M-current inhibition involves a diffusible element (Selyanko et al. 1992). Since M-type K+ channels in isolated patches are inhibited by Ca2+ with an EC50 of 100 nM (Selyanko & Brown, 1996), one obvious candidate for this diffusible cascade is InsP3 generated by muscarinic activation of PLC, which would release Ca2+ from intracellular stores to directly inhibit M-channels. Release of Ca2+ from intracellular stores appears to mediate muscarinic M-current inhibition in amphibian sympathetic neurones (Kirkwood et al. 1991). There are, however, a number of unanswered questions associated with this scheme. Firstly, it is not known whether concentrations of muscarinic agonist which activate PLC in SCG neurones also mobilise intracellular calcium (Ca2+i); indeed, previous studies of rat voltage-clamped SCG neurones have failed to demonstrate an increase in Ca2+i after muscarinic receptor activation (Wanke et al. 1987; Beech et al. 1991; Marsh et al. 1995; Cruzblanca et al. 1998). Also, previous measurements of muscarinic receptor activation of PLC (Bone et al. 1984; Horwitz et al. 1985; Ramcharan & Matthews, 1996) were carried out on intact ganglia, which contain non-neuronal as well as neuronal cells, so there is the possibility that some part of the response involved a non-neuronal component, since glial PLC can be stimulated by muscarinic agonists (Pearce et al. 1988; Konou et al. 1994; Shao & McCarthy, 1995). Finally, rat superior cervical ganglion neurones may express M3 as well as M1 receptors, since mRNA species encoding both subtypes have been detected by in situ autoradiography (Brown et al. 1995), and it is unclear whether the M3 receptor complement may contribute to muscarinic PLC activation (in other systems, M3 receptors do couple to PLC; Caulfield, 1993). Interest in the nature of the diffusible second messenger mediating muscarinic IK(M) inhibition has recently been further stimulated by the observation of Cruzblanca et al. (1998) that bradykinin (which also inhibits IK(M) in SCG neurones: Jones et al. 1995) does elevate Ca2+i in these neurones, and that it is this increase in Ca2+i (via PLC activation and InsP3 generation) which transduces M-current inhibition by bradykinin. In contrast, Cruzblanca et al. found that muscarinic IK(M) inhibition was not associated with increased Ca2+i. In the present work we show that the muscarinic receptor agonist oxo-M does activate PLC in SCG neurones, and that it does increase Ca2+i, provided that the calcium stores are pre-loaded and/or sensitised by depolarising the neurones to open voltage-gated calcium channels. However, we confirm the report of Cruzblanca et al. (1998) that muscarinic M-current inhibition persists under conditions in which the muscarinic Ca2+i rise is greatly reduced or abolished, and hence that this is unlikely to mediate M-current inhibition. METHODS The extracellular solution (modified Krebs solution) for cytoplasmic calcium measurements and electrophysiology contained (mm): NaCl 120; NaHCO3 23; Hepes 5; glucose 11; KCl 3; MgCl2 1.2; CaCl2 2.5; tetrodotoxin (TTX; Alomone Labs, Jerusalem, Israel) 0.0005. Electrophysiological recordings and ratiometric determination of intracellular calcium Current recording and microfluorimetric fura-2 estimations of Ca2+i were carried out simultaneously on single neurones. Neurones (2–5 days in culture) were loaded with fura-2 AM (Molecular Probes, 1–2 μm) for 1 h in culture medium at 37°C in 5 % CO2, followed by washing for 30 min in modified Krebs solution at 34°C. Changes in Ca2+i were estimated from the ratio of fura-2 fluorescence (at 500 nm) with alternate excitation at wavelengths of 340 and 380 nm, generated by a Spectramaster monochromator (Life Science Resources, Cambridge, UK). Wavelength switching was at a frequency of 0.5–1 Hz. Intensity of illumination was minimised to reduce bleaching of fura-2, while maintaining adequate levels of signal. Fura-2 images for digital analysis were generated using a system based on an Olympus BX50WI microscope coupled to an image intensifier and CCD camera, and were analysed with MiraCal software (Life Science Resources). On-cell calibrations of fura-2 ratios (R) for a range of Ca2+i concentrations were done using modified patch pipette solutions in the 'whole-cell' mode, essentially as described by Trouslard et al. (1993), using calcium standards from Molecular Probes. Best-fit parameters of the equation were Kd (fura-2 dissociation constant) = 224 nM; β= 3.7; Rmax (ratio at saturating calcium concentration) = 4; Rmin (minimal ratio value at zero calcium concentration) = 0.4. To allow estimates to be made of the concentrations of fura-2 achieved after the fura-2 AM loading procedure, we generated an intensity-fura-2 concentration calibration line, determined by incorporating varying concentrations of fura-2 potassium salt in the pipette solution, and monitoring emission intensity at 360 nm (isobestic point for fura-2). Emission intensity gradually increased after breaking through into the 'whole-cell' mode, reaching a plateau at 15–20 min after breakthrough, when measurements were made. Estimated cytoplasmic fura-2 concentrations were < 100 μm. The intrapipette solution for electrophysiological measurements contained (mm): potassium acetate 60; KCl 60; MgCl2 2.5; BAPTA 10; Hepes 30; adjusted to pH 7.4 with KOH. Internal and external solutions had osmolarities of about 290 mosmol l−1. Fura-2 AM was prepared as a 1 mm stock in dimethyl sulfoxide (DMSO) and was added to neurones at the final desired concentration in culture medium (see below). Neuronal culture Primary cultures of Sprague-Dawley rat (age 15–21 days) SCG principal neurones were prepared and maintained as described previously (Caulfield et al. 1994). Animals were killed in accordance with Schedule 1 of the Animals (Scientific Procedures) Act (UK). The method of killing consisted of hypoxia by exposing the rats to a rising concentration of carbon dioxide. For electrophysiological and Ca2+i measurements cells were plated on laminin-coated (10 μg ml−1) glass coverslips. For PLC turnover measurements, cells were plated in 96-well plates, also pre-coated with laminin. For recordings, coverslips were placed in a bath (volume about 1 ml) and perfused with modified Krebs solution (34°C) at a rate of about 0.5 ml s−1. Drugs were applied in the perfusate, and complete solution exchange was achieved in under 2 s. Neurones were voltage clamped with fire-polished borosilicate electrodes (2–4 MΩ) back-filled with amphotericin (0.1 mg ml−1 in 0.1 % DMSO), to allow perforated patch recording (Horn & Marty, 1988; Rae et al. 1991) using an Axopatch 1D amplifier (Axon Instruments) at a sampling rate of 4 kHz (filter 0.2 kHz). Access resistances were < 30 MΩ. Neurones were initially clamped at −60 mV, and then depolarised to −25 mV to pre-activate IK(M). IK(M) was measured as the slowly developing deactivation of the outward current during a 1 s step to a command potential of −55 mV (Caulfield et al. 1994). Data were collected and analysed using pCLAMP 6 software (Axon Instruments). Measurement of PLC activation The method of Downes & Stone (1986) was used to determine PLC activity in neurones. It relies on the level of an intermediate in the resynthesis of phospatidylinositol, CMP-PA, which accumulates during PLC stimulation as a result of the inhibition of the inositol monophosphate phosphatase and subsequent depletion of cytoplasmic inositol by 10 mm LiCl (del Río et al. 1996). Each data point was obtained from tissue yielded by an individual ganglion. Radiolabelling with 100 μCi ml−1[3H]-cytidine (ICN, 23 Ci mmol−1) was performed in tissue culture medium containing 10 mm LiCl at 37°C for 1 h. Agonist stimulation of CMP-PA accumulation was terminated by adding trichloroacetic acid (TCA, 5 % final concentration). Supernatants were discarded, and lipids extracted from the TCA-insoluble material by incubation with 750 μl of chloroform:methanol:11 M HCl (40:80:1) at 4°C for 20 min, and phase separation upon addition of 250 μl of chloroform and 400 μl of 0.1 M HCl. [3H] content of aliquots of the hydrophobic phase was determined by liquid scintillation counting. It has previously been demonstrated that CMP-PA is the only [3H]-cytidine containing lipid accumulated under these conditions (Godfrey, 1989). Accumulation of CMP-PA in the presence of Li+ is an advantageous measure of phosphoinositide turnover in systems (such as this) in which tissue availability is limiting (each data point corresponds to 1000-5000 SCG neurones), making measurements of inositol phosphates impractical. Furthermore, it yields a very favourable signal-to-noise ratio, enabling detection of phosphoinositide turnover by microscopic techniques. In situ CMP-PA localisation Autoradiographic localisation of CMP-PA in SCG cultures was carried out as described by Bevilacqua et al. (1994), with minor modifications. Prior to [3H]-cytidine (5 μCi ml−1) labelling in the presence of 10 mm LiCl, cultures were pre-incubated with 50 mm hydroxyurea plus 5 μg ml−1 actinomycin D for 30 min. After agonist stimulation, cultured cells were fixed with 2 % paraformaldehyde plus 2.5 % glutaraldehyde, in phosphate-buffered saline, followed by RNase and DNase treatment, in the presence of 0.005 % saponin and 3 % polyethyleneglycol and post-fixation with 2 % paraformaldehyde plus 2.5 % glutaraldehyde, in phosphate-buffered saline. The treatment of neurones with actinomycin D and hydroxyurea followed by permeabilisation with RNase and DNase greatly reduces incorporation of [3H]-cytidine into nucleic acids, which would otherwise interfere with in situ autoradiographic localisation of [3H]-CMP-PA. Slides were coated with Ilford K5 autoradiographic emulsion, and exposed for 9–12 days prior to developing, staining with Richardson's Blue and mounting. In all cases data are reported as means ±s.e.m. of the indicated number (n) of replicates. RESULTS Muscarinic stimulation of CMP-PA accumulation In the presence of 10 mm LiCl, the muscarinic agonist oxo-M (10 μm; 15 min incubation) increased CMP-PA accumulation in SCG neurone cultures by 23-fold over background levels recorded in the absence of LiCl (Fig. 1A). Pre-incubation with 10 mm inositol for 48 h completely blocked the oxo-M effect, verifying the use of CMP-PA accumulation as a specific measure of phosphoinositide turnover. Accumulation of CMP-PA stimulated by this concentration of oxo-M had a lag phase of approximately 2 min and was maximal at 15 min (Fig. 1B), so this incubation time was used for subsequent experiments. (The initial lag phase represents the period over which cytoplasmic inositol becomes depleted, while maximal accumulation reflects inactivation of PLC or of CMP-PA synthesis after a prolonged stimulation.) The threshold concentration for oxo-M stimulation of PLC was 0.1–1 μm, with an EC50 value of 3.6 μm (Fig. 1C; pEC50 5.45 ± 0.15). Responses were close to maximum at 100 μm oxo-M. Because of the variability in the yield of neurones between cultures (and therefore the magnitude of the CMP-PA accumulation), responses were normalised with respect to the response obtained with 10 μm oxo-M (the calibrating concentration of oxo-M). Figure 1Open in figure viewerPowerPoint Characterisation of oxo-M-induced CMP-PA accumulation in SCG neuronal cultures A, oxo-M (10 μm, 15 min) stimulated CMP-PA accumulation only in the presence of 10 mm LiCl, and this effect was antagonised by 48 h pre-incubation with 10 mm inositol. B, oxo-M (10 μm) induced CMP-PA accumulation was detectable after 5 min incubation with agonist and reached a maximum at 15-30 min. C, oxo-M (15 min) stimulates the accumulation of CMP-PA in a dose-dependent manner (concentration range 0.1–100 μm). D, concentration-response curve for the effect of bradykinin on CMP-PA accumulation. In this case stimulations lasted 30 min and responses are expressed as percentage of the 30 min stimulation with 10 μm oxo-M performed in the same series of experiments (concentration range 1 nM to 10 μm). In all cases data are means ±s.e.m. of 4–6 independent determinations. Typically control values ranged from 100 to 400 c.p.m. and maximal stimulation from 2500 to 12000 c.p.m. Because of the variability in the yield of neurones between cultures (and therefore the magnitude of the [3H]-CMP-PA accumulation), responses are expressed as a percentage of the 10 μm oxo-M-induced stimulation of CMP-PA accumulation (the calibrating concentration of oxo-M) over a 15 min period, unless otherwise stated. Bradykinin stimulation of CMP-PA accumulation Bradykinin evoked a dose-dependent accumulation of CMP-PA in the presence of 10 mm LiCl, which was much smaller than that observed with the muscarinic agonist (Fig. 1D). The maximally effective concentration of bradykinin was 1 μm; at this concentration CMP-PA accumulation was 13.4 ± 4 % of that evoked with the calibrating concentration of oxo-M. Effect of the aminosteroids U73122 and U73343 on oxo-M stimulated PLC activity It has previously been shown that bradykinin-induced inhibition of M-current can be prevented by the aminosteroid U73122 (a putative selective PLC antagonist) but not by its structural analogue U73343, while muscarinic inhibition of M-current is insensitive to U73122 (Cruzblanca et al. 1998). We have tested the ability of these aminosteroids to inhibit PLC activation by oxo-M. Figure 2 shows the dose dependence of the inhibition of CMP-PA accumulation by U73122 and U73343. While U73122 failed to inhibit CMP-PA accumulation in a statistically significant manner at concentrations ≤ 10 μm, the inactive structural analogue U73343 (10 μm) caused an 80 % inhibition of CMP-PA accumulation (P < 0.05, significantly more effective than U73122 at 10 μm, Student's t test). Higher concentrations of both aminosteroids led to a recovery of CMP-PA accumulation. Figure 2Open in figure viewerPowerPoint Sensitivity of PLC activity to the aminosteroids U73122 and U73343 SCG cultures were pre-incubated with a range of concentrations of the aminosteroids U73122 (▴) or U73343 (▪) for 45 min prior to stimulation with 10 μm oxo-M (the calibrating concentration of oxo-M). U73122 and U73343 were prepared as 1.5 mm stock in ethanol and were added to neurones at the final desired concentration in culture medium, maintaining a constant extracellular concentration of ethanol of 1.66 % for all data points. *P < 0.05, Student's t test. Neuronal localisation of CMP-PA accumulation Autoradiographic detection of [3H]-CMP-PA after 10 μm oxo-M-induced stimulation in the presence of Li+ demonstrated an inositol-sensitive signal evident in SCG neurones. In contrast, the signal was absent in glial cells (Fig. 3). In situ localisation of bradykinin-stimulated PLC activity could not be performed due to the small magnitude of the CMP-PA accumulation evoked by bradykinin in SCG neurones. Figure 3Open in figure viewerPowerPoint Autoradiographic in situ localisation of CMP-PA in SCG cultures [3H]-Cytidine incorporation into CMP-PA is stimulated by 10 μm oxo-M (15 min) in the presence of 10 mm LiCl (A and B; bright- and dark-field images of the same field of cells) and autoradiographic grains are clearly evident in SCG neurones (arrowed), but not in glia. Background grain levels are shown in preparations pretreated with 10 mm inositol for 48 h (C and D; bright- and dark-field images of the same field of cells). Scale bar in A represents 50 μm and applies to all panels. Role of Ca2+ influx in PLC activation Oxo-M (10 μm) can evoke nicotinic currents in sympathetic neurones (Xian et al. 1994), and the influx of calcium through nicotinic receptor channels can significantly elevate intraneuronal calcium (Trouslard et al. 1993). Since muscarinic receptor stimulation of PLC can be markedly enhanced by procedures which increase intracellular calcium (Baird & Nahorski, 1989; Fisher et al. 1989; del Río et al. 1994), we tested whether nicotinic calcium elevation may have contributed to the stimulatory effect of oxo-M on PLC activity. In the presence of trimetaphan (30 μm), a competitive, reversible antagonist of nicotinic receptors, 10 μm oxo-M stimulated PLC by 88 ± 7 % (n= 4), which was not significantly different from the effect of oxo-M alone (P > 0.05, ANOVA). A similar lack of effect of trimetaphan was seen with 100 μm oxo-M, where PLC stimulations with or without antagonist were 115 ± 3 % (n= 4) and 136 ± 7 % (n= 4) of the 10 μm oxo-M effect, respectively (P > 0.05, ANOVA). Thus, oxo-M stimulation of PLC (in the concentration range we used) does not involve any significant nicotinic receptor component. We also tested the effect of 40 mm KCl-induced depolarisation (which causes a substantial transient and non-deactivating increase in Ca2+i; data not shown) on the basal and oxo-M-evoked accumulation of CMP-PA. In neither case did the presence of 40 mm KCl enhance the accumulation of CMP-PA in a statistically significant manner (P > 0.05, ANOVA). Hence, the effect of oxo-M was unlikely to be secondary to a rise in Ca2+i. Muscarinic receptor pharmacology Oxo-M-induced stimulation of PLC in SCG neurones was antagonised by the partially selective muscarinic antagonists pirenzepine and himbacine. In dose ratio experiments with 100 nM pirenzepine and 1 μm himbacine, the concentration- response curves for oxo-M were shifted to the right by factors of 10 and 13, respectively (Fig. 4). There was no evidence of a change in the slope of the concentration- response curve for oxo-M in the presence of either antagonist, suggesting that the Gaddum-Schild equation (KB=[B]/(DR - 1), where KB is the apparent dissociation constant, [B] is antagonist concentration and DR is dose ratio) can be applied to allow calculation of antagonist apparent pKB values. pKB values were 7.9 ± 0.4 for pirenzepine and 7.1 ± 0.6 for himbacine. Figure 4Open in figure viewerPowerPoint Characterisation of the muscarinic receptor type that mediates oxo-M-induced stimulation of PLC in SCG neuronal culture Pre-incubation (1 h) with 100 nM pirenzepine (left; ▴) or 1 μm himbacine (right; ▴) shifts the control oxo-M curve (•) rightwards without any apparent reduction in maximum response. Himbacine at 100 nM (right, ▾) did not significantly reduce the oxo-M responses. Applying the Gaddum-Schild equation, pKB values for pirenzepine and himbacine were 7.95 and 7.1, respectively, indicating that the PLC response is mediated by the M1 muscarinic receptor subtype. Depolarisation to pre-activate IK(M) increases Ca2+i and reveals an elevation of Ca2+i in response to muscarinic agonist In previous voltage-clamp studies on muscarinic mobilisation of Ca2+i in SCG neurones, the cells were clamped at around resting potential (i.e. −60 mV); under these conditions, no elevation in Ca2+i was observed following application of muscarinic agonists (Marsh et al. 1995; Cruzblanca et al. 1998). However, the classical procedure for recording IK(M) involves pre-depolarising the neurone under voltage clamp to around −25 mV (Brown & Adams, 1980). We therefore tested whether oxo-M affected Ca2+i in neurones depolarised to −25 mV. On depolarisation to −25 mV, Ca2+i increased from a mean basal value of 89.4 ± 12.9 nM to 251 ± 40.9 nM (n= 9; P < 0.05, ANOVA; Fig. 5); this level was sustained throughout the depolarisation. Oxo-M at 1 μm, a concentration of agonist which inhibits IK(M) by about 70 % (Haley et al. 1998), further increased Ca2+i in each pre-depolarised neurone tested by approximately 25 % (mean increase 67 ± 15 nM; n= 9). The oxo-M-induced further Ca2+i increase was not maintained, fading to pre-agonist levels in about 40 s (Fig. 4). In view of the transient nature of this response, it was measured as the highest Ca2+i achieved within 90 s of agonist addition. It ought to be noted that this small effect of oxo-M on Ca2+i was not always reproduced when the agonist was repeatedly applied to the same cell, or to different cells on the same coverslip. Consequently, agonist applications were made only once for any given cell on a given coverslip, and control responses were obtained throughout the whole experimental series, so that the mean control values presented can be validly compared to each of the test groups, which were likewise randomised throughout the experiments. Figure 5Open in figure viewerPowerPoint Muscarinic receptor activation increases Ca2+i following neuronal depolarisation A, representative fura-2 traces showing Ca2+i levels in two neurones, one voltage clamped at −60 mV, the other depolarised to −25 mV about 20 s after the start of the sweep. Depolarisation is associated with a substantial increase in Ca2+i. Application of oxo-M (indicated by the bar) does not change Ca2+i in the neurone held at −60 mV, but induces a further transient increase in Ca2+i in the neurone held at −25 mV. The mean absolute Ca2+i in the presence of agonist was 313 ± 50 nM (n= 9), while the mean increase in Ca2+i upon agonist addition was 67 ± 16 nM. B, IK(M) deactivation relaxations in the neurone held at −25 mV in A, evoked by stepping for 1 s to −55 mV. Traces are shown before (control) and 90 s after application of oxo-M, which has reduced both the standing outward current, and the relaxation during the hyperpolarising voltage step. M-current inhibition developed slowly, reaching maximum level 30 s after oxo-M application, and persisted at this level as long as oxo-M was present in the extracellular medium (> 10 min). The calcium mobilisation response was not observed in cells loaded with higher fura-2 concentrations (intracellular fura-2 concentration approximately 0.3–1 mm). However, these cells showed normal inhibition of M-current over a range of agonist concentrations (data not shown). In agreement with Cruzblanca et al. (1998) we could also observe Ca2+ release responses to bradykinin (1 μm) in SCG neurones voltage clamped at −20 mV in perforated patch mode. Clear Ca2+ release responses were observed in six out of nine cells studied. These responses were always equal to or larger than parallel Ca2+ release responses to oxo-M (10 μm) (bradykinin, 53 ± 23 nM, n= 6; oxo-M, 41 ± 6 nM, n= 2). This series of Ca2+ release determinations was performed independently from those described earlier in this section. M-current and muscarinic M-current inhibition are not affected by changes in Ca2+i Most voltage-activated calcium channel blockers reduced both the depolarisation-induced rise in Ca2+i and the oxo-M-induced Ca2+i rise in depolarised neurones. However, it was not feasible to completely block the Ca2+i rise evoked by depolarisation whilst measuring M-current inhibition, as most of the calcium channel blockers tested (ω-conotoxins GVIA and MVIIC; 100 μm Cd2+; 3 mm Ni2+) themselves reduced IK(M) by at least 20 % (data not shown). Nifedipine (1 μm) was the only calcium channel blocker which did not affect M-current or the Ca2+ release response to oxo-M (Fig. 6), although it decreased Ca2+i in all depolarised neurones tested (mean decrease 52 ± 6 nM, n= 4); this change in Ca2+i in individual neurones was not associated with a change in IK(M). Figure 6Open in figure viewerPowerPoint Nifedipine reduces Ca2+i in depolarised neurones, does not abolish the Ca2+i rise with oxo-M, and does not modify M-current or its inhibition by oxo-M A, a fura-2 trace shows Ca2+i in a voltage-clamped neurone stepped to −25 mV about 10 s after the beginning of the recording (evident as the increase in Ca2+i at that point). Application of nifedipine reduces the Ca2+i level, and addition of oxo-M in the presence of nifedipine still evokes an increase of Ca2+i of the same magnitude as in the absence of nifedipine. Absolute Ca2+i in the presence of nifedipine plus oxo-M was 210 ± 78 nM (n= 9), and the transient Ca2+ release response upon application of oxo-M was 47 ± 2 nM (n= 4), which was not statistically significantly different from the response in the absence of nifedipine. B, current
Referência(s)