Muscarinic Receptor Binding Sites of the M4 Subtype in Porcine Lung Parenchyma

Abstract: 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 M₄ subtype by careful comparison of antagonist affinities. We characterized the binding of ³H]quinuclidinyl benzilate (³H]QNB) to muscarinic receptors in cell membranes from lung parenchyma of 2–8 week old pigs in comparison to cloned human M₃ and M₄ receptors expressed in COS cells, to M₂ in rat atria and to M₄ in bovine adrenal medulla. In porcine lung, ³H]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 m₄ (r=0.996). A series of 10 blockers showed affinities closely matching reported values for M₄ 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 M₃ or with rat atria expressing the M₂ subtype. We conclude that pig lung muscarinic receptor binding sites most closely resemble the M₄ subtype, in contrast to the M₃ subtype typical of large airways in this species.