Muscarinic M1receptors activate phosphoinositide turnover and Ca2+mobilisation in rat sympathetic neurones, but this signalling pathway does not mediate M‐current inhibition

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 pK_B (—log of the apparent dissociation constant) of 7.9 ± 0.4, while himbacine (1 μm) shifted the curve by a factor of about 13 (pK_B∼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.