Muscarinic M-1 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, phosphoinositid e 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 [H-3]-cytidine monophosphate phosphatidate (CMP-PA) after incubation with [H-3]-cytidine in the presence of Lif. The muscarini c agonist oxotremorine methiodide (oxo-M) stimulated PLC in a dose-dependen t manner with an EC50 of approximately 3.5 mu M. 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 pK(B) (-log of th e apparent dissociation constant) of 7.9 +/- 0.4, while himbacine (1 mu M) shifted the curve by a factor of about 13 (pK(B) similar to 7.1 +/- 0.6). T his indicates involvement of the M-1 muscarinic receptor in this response. 4. The accumulation of CMP-PA was localised by in situ autoradiography to S CG 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 ( InsP(3))-sensitive stores was determined by fura-2 microfluorimetry of SCG neurones voltage clamped in perforated patch mode. Oxo-M failed to evoke in tracellular Ca2+ (Ca-1(2+)) mobilisation in SCG neurones voltage clamped at -60 mV, but produced a significant Ca-1(2+) rise (67 +/- 15 nM, n = 9) in cells voltage clamped at -25 mV. 6. Thapsigargin (0.5-1 mu M) caused a 70% inhibition of the oxo-M-induced C a-1(2+) 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 InsP(3)-sensitive calcium mobilisation in M-current inhibition.