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J Physiol Volume 520, Number 1, 101-111, October 1, 1999
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The Journal of Physiology (1999), 520.1, pp. 101-111
© Copyright 1999 The Physiological Society

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, Jorge A. Bevilacqua *, Stephen J. Marsh ¹, Pamela Halley and Malcolm P. Caulfield

Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK, * ICBM-Facultad de Medicina, Universidad de Chile, Independencia 1027, cc 70079, Santiago, Chile and ¹Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK


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.


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.


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.


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.


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.


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.


Our results do not support the involvement of InsP3-sensitive calcium mobilisation in M-current inhibition.


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