Muscarinic receptor binding sites of the M4 subtype in porcine lung parenchyma

Muscarinic acetylcholine receptors regulate distal airway resistance and secretion. The subtype expressed in the lung in different species remains uncertain. It has recently become possible to identify the M-4 subtype by careful comparison of antagonist affinities. We characterized the binding of [H-3]quinuclidinyl benzilate ([H-3]QNB) to muscarinic receptors in cell membranes from lung parenchyma of 2-8 week old pigs in comparison to cloned human M-3 and M-4 receptors expressed in COS cells, to M-2 in rat atria and to M-4 in bovine adrenal medulla. In porcine lung, [H-3]QNB bound with high affinity (K-d = 95+/-9pM) to a single homogeneous population of muscarinic receptor sites (B-max = 340+/-10 fmol/mg protein). Competition studies showed that the affinity (expressed as pK(i)) of 3 selective blockers was in close agreement between pig lung and cloned human m(4) (r=0.996). A series of 10 blockers showed affinities closely matching reported values for M-4 receptors of the adrenal medulla (r=0.965). Conversely, affinity values in porcine lung differed significantly (P< 0.05, t-test) from those determined in parallel with either human cloned M-3 or with rat atria expressing the M-2 subtype. We conclude that pig lung muscarinic receptor binding sites most closely resemble the M-4 subtype, in contrast to the M-3 subtype typical of large airways in this species.