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 maintained 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 (Kd = 70 pM) to a homogeneous population of receptors on intact neurons (320 fmol/mg cellular protein). Displacement of the binding of [3H]NMS by pirenzepine indicated the presence of heterogeneous sites (81% high affinity sites, Kh = 51 nM, K1 = 1.5 microM); AF-DX 116 showed the opposite selectivity (15% high affinity sites, Kh = 56 nM, K1 = 1.3 microM). The dopamine agonist SKF-38393 (1 microM) enhanced the accumulation of cyclic adenosine monophosphate (AMP) in these cultures 2.5-fold; addition of carbachol reduced cyclic AMP levels by 30% (EC50, 1.7 microM). In the presence of 1 mM lithium, carbachol stimulated the accumulation of inositol monophosphate 5-fold (EC50, 61 microM). Both responses were antagonized by pirenzepine (apparent Ki of 23 nM for the phosphoinositide response and 200 nM for the cyclic AMP response) and AF-DX 116 (apparent Ki 540 nM and 160 nM, respectively). In binding studies on brainstem cultures, AF-DX 116 indicated the presence of two sites of approximately equal abundance (Kh = 170 nM, K1 = 2.9 microM); data for pirenzepine were adequately fit by a one-site model (Kd = 630 nM).(ABSTRACT TRUNCATED AT 250 WORDS)