Cytochalasin Modulation Of Nicotinic Cholinergic Receptor Expression And Muscarinic Receptor Function In Human Te671/Rd Cells: A Possible Functional Role Of The Cytoskeleton

Abstract

Abstract: Previous studies have shown that cells of the TE671/RD human clonal line express muscleâ€type nicotinic acetylcholine receptors (nAChR) and m3â€type muscarinic acetylcholine receptors (mAChR) whose numbers and function are regulated by agonist treatment and second messenger modulation. Here we show that cytochalasin treatment, which causes disruption of actin networks, induces marked changes in the numbers and distribution of nAChR, but not mAChR. Moreover, whereas cytochalasin treatment fails to alter nAChR function significantly, it acutely potentiates mAChRâ€mediated phosphoinositide hydrolysis. Treatment of TE671/RD cells with different cytochalasin analogues (rank order efficacy at 5 μg/ml is H \u3eJ = B = C = D\u3eA = E) produces a twoâ€to fourfold increase in numbers of membraneâ€bound nAChR (Smax in units of specific 125lâ€labeled αâ€bungarotoxin binding per milligram of membrane protein). nAChR upâ€regulation is evident after 1–2 days of cytochalasin B exposure, is maximal after 3–6 days of drug treatment, and is dominated by an approximately 10â€fold increase (per cell) in an intracellular nAChR pool. Cytochalasinâ€induced nAChR upâ€regulation is similar in magnitude to, but not additive with, upâ€regulation of nAChR following chronic exposure to nicotine or phorbol ester. Northern blot analysis shows a fourâ€to fivefold coordinate increase in levels of mRNA that encode nAChR α, β, γ, or θ subunits in cytochalasinâ€treated cells, suggesting that nAChR upâ€regulation has a possible transcriptional basis. Studies done using a 86Rb+ efflux assay indicate that cytochalasin treatment has no significant effect on nAChR function. By contrast, cytochalasin treatment has no effect on the numbers of mAChR as assessed by binding studies with the radioantagonist 3Hâ€labeled quinuclidinyl benzilate, but it induces marked enhancement of carbacholâ€stimulated, but not basal, phosphoinositide hydrolysis. These studies suggest that presumed modulation by cytochalasin treatment of cytoskeletal microfilament integrity can differentially influence expression and function of mAChR (a prototype of the metabotropic receptor superfamily) and nAChR (a prototype of the ligandâ€gated ionâ€channel superfamily). The results also suggest possible new roles for the cytoskeleton in regulation of membrane receptor expression, function, and cross talk. Copyright © 1993, Wiley Blackwell. All rights reserve