Cyclic AMP-dependent Mechanism Regulates Acetylcholine Receptor Function on Bovine Adrenal Chromaflin Cells and Discriminates between New and Old Receptors

Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that mediate release of catecholamines from the cells in response to synaptic input, and resemble neuronal AChRs in pharmacology and antigenic profile. Results presented here show that a cAMP-dependent process enhances the function of adrenal chromaffin AChRs as a population in the plasma membrane. This was demonstrated by showing that cAMP analogues cause specific increases both in the level of nicotine-induced catecholamine release from the cells and in the level of the nicotineinduced conductance change occurring in the cells. Neither de novo synthesis of receptors nor transport of preexisting intracellular receptors to the plasma membrane is necessary for the enhancement. The responsiveness of AChRs to regulation by the cAMP-dependent process appears to depend on the length of time the receptors have been on the cell surface. AChRs newly inserted into the plasma membrane generate a greater nicotinic response than do older AChRs and, unlike older AChRs, their response to agonist is not enhanced after treatment of the cells with cAMP analogues. The findings indicate that the AChRs and/or associated components undergo a maturation in the plasma membrane that alters their function and their regulation by secondary messenger systems. N 'EUROTRANSMITTER receptors in the postsynaptic membrane mediate synaptic transmission at all chemical synapses. Because of this central role, the receptors represent a likely site at which regulatory mechanisms might act to modulate synaptic transmission. Recent studies with nicotinic acetylcholine receptors (AChRs) t on chick ciliary ganglion neurons have indicated that a cAMPdependent mechanism enhances the acetylcholine (ACh) sensitivity of the cells (Margiotta et al., 1987a). The enhancement appears to represent an increase in the number of functional AChRs on the neurons since no changes that could account for the effect are observed in AChR single channel proPerties. The increased ACh sensitivity, however, occurs in the absence of protein synthesis, implying that it represents a conversion of preexisting nonfunctional AChRs to functional ones (Margiotta et al., 1987a). The source of the nonfunctional AChRs has not been determined. It could derive either from a substantial population of nonfunctional AChP, s thought to be present in the plasma membrane (Margiotta et al., 1987b) or from a large population of intracellu1. Abbreviations used in this paper: ACh, acetylcholine; AChR, nicotinic acetylcholine receptor; ~t-Bgt, a-bungarotoxin; BrACh, bromoacetylcholine; DTNB, 5,5-dithio-bis(2-nitrobenzoate); 8-Br-cAMP, 8-bromoadenosine 3',5'-cyclicmonophosphate; 3H-NE, 3H-norepinephrine; IBMX, 3-isobutyl-l-methylxanthine; n-Bgt, neuronal bungarotoxin. lar AChRs known to be contained in the neurons (Stollberg and Berg, 1987). The possibility that the number of functional AChRs on cells can be increased by a cAMP-dependent process suggests ways in which cell-cell interactions might regulate synaptic signaling. Regulation of this type apparently does not apply to AChRs in vertebrate skeletal muscle or electric organ. Activation of cAMP-dependent processes in muscle reduces ACh sensitivity in a manner consistent with the receptors becoming more susceptible to agonist-induced densensitization (Albuquerque et al., 1986; Middleton et al., 1986). Reconstitution experiments have directly demonstrated that cAMP-dependent phosphorylation of electric organ AChRs enhances agonist-induced desensitization of the receptors (Huganir et al., 1986). Neuronal AChRs, however, are known to differ from muscle AChRs in certain pharmacological, structural, and regulatory aspects (Boulter et al., 1986, 1987; Jacob and Berg, 1987, 1988; Patrick and Stallcup, 1977a, b; Whiting and Lindstrom, 1986, 1987). The ability to recruit functional AChRs from a nonfunctional receptor pool by a cAMP-dependent process could provide neurons a useful flexibility in detecting synaptic signals that would be unnecessary at the neuromuscular junction. AChRs on bovine adrenal chromaffin cells appear to be of the neuronal type. They resemble neuronal AChRs in single © The Rockefeller University Press, 0021-9525/88/09/1157/9 $2.00 The Journal of Cell Biology, Volume 107, September 1988 1157-1165 1157 on S etem er 3, 2016 D ow nladed fom Published September 1, 1988