Muscarinic M1 receptors activate phosphoinositide turnover and Ca2+ mobilisation in rat sympathetic neurones, but this signalling pathway does not mediate M-current inhibition.
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 microM. 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 microM) shifted the curve by a factor of about 13 (pKB approximately 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 microM) 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.
del Río
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Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK. rmgzeda@ucl.ac.uk
Bevilacqua
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Marsh
S J
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Halley
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Caulfield
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MP
D000465:Algorithms; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D003568:Cytidine Monophosphate / Q000378:metabolism; D004594:Electrophysiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D018721:Muscarinic Agonists / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D010095:Oxotremorine / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism* / Q000502:physiology; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D013564:Sympathetic Nervous System / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism
D018721:Muscarinic Agonists; D010716:Phosphatidylinositols; D015221:Potassium Channels; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D010095:Oxotremorine; D010738:Type C Phospholipases; D003568:Cytidine Monophosphate; D002118:Calcium