Muscarinic receptors and second-messenger responses of neurons in primary culture

The coupling of muscarinic receptors to second messenger responses was investigated in primary cultures of neurons from the fetal mouse brain. Neurons were mainained in monolayer culture, in serum-free medium; immunocytochemical studies found these cultures to be nearly exclusively neuronal. In striatal cultures, [³H]N-methylscopolamine (NMS) bound specifically and with high affinity (K_d= 70 pM) to a homogenous population of receptors on intact neurons (320 fmol/mg cellular protein). Displacement of the binding of [³H]NMS by pirenzepine indicated the presence of heterogeneou sites (81% high affinity site, K_h= 51 nM, K₁= 1.5 μM); AF-DX 116 showed the oppisite selectivity (15% high affinity sites, K_rmh= 56 nM, K₁= 1.5 μM). The dopamine agonist SKF-3893 (1 μM) enhanced the accumulation of cylic adenosine monosphosphate (AMP) in these cultures 2.5-fold; addition of carbachol reduced cyclic AMP levels by 30% (EC₅₀, 1.7 μM). In the presence of 1 mM lithium, carbachol stimulated the accumulation of inositol monophosphate 5-fold (EC₅₀, 61 μM). Both responses were antagonized by pirenzepine (apparent K₁ of 23 nM for the phosphoinositide response and 200 nM for the cyclic AMP response) and AF-DX 116 (apparent K_i 540 nM and 160 nM, respectively). In binding studies on braiinstem cultures, AF-DX 116 indicated the presence of two sites approximately equal abundance (K_h = 170 nM, K_{1= 2.9 υM}); data for pirenzepine were adequately fit by one-site model (K_d= 630 nM. The efficacy of oxotremorine (compared to oxotremorine-M) in stimulating the phosphoinositide response was equally low in striatal and cortical cultures (0.07), but significantly greater in brainstem cultures (0.26). These studies suggest that the striatal phosphoinositide and cyclic AMP responses were mediated by M₁ and M₂ receptors, respectively, while the brainstem phosphoinositide response was mediated by M₃ receptors.