Abstract
BackgroundNicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine.MethodsRat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted.ResultsPositive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium.ConclusionsRat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.
Highlights
Nicotinic acetylcholine receptors have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events
Rat alveolar macrophages (AM) are equipped with modulatory Nicotinic acetylcholine receptors (nAChR) with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system
Rat alveolar macrophages constitutively express nAChR subunits a9, a10 and b2, but not a7 RT-PCR analysis of mRNA isolated from rat bronchoalveolar lavage (BAL) cells revealed expression of nAChR subunits a2, a3, a5, a9, a10, b1, and b2
Summary
Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. It has been demonstrated that TNFa production and release from peritoneal macrophages can be largely inhibited by neurally released ACh thereby attenuating systemic inflammatory responses. This physiological mechanism has been subunit is one of 9 different known ligand-binding a subunits (a1-a7 and a9-a10) that assemble to homoor heteropentamers, partially with additional participation of b subunits, to form a functional nAChR. All these receptors are ligand-gated cation channels, and they are distinct from each other with respect to ligand affinity and to preference for mono- or divalent cations [4]. This allows for selective pharmacological intervention and therapeutic use
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