Role of Acetylcholine Receptors and Dopamine Transporter in Regulation of Extracellular Dopamine in Rat Carotid Body Cultures Grown in Chronic Hypoxia or Nicotine

Abstract

Using dissociated rat carotid body (CB) cultures, we compared levels of extracellular dopamine (DA) around oxygen-sensitive glomus cells grown for -~12 days in normoxia (Nox; 20% 02), chronic hypoxia (CHox; 6% 02), or chronic nicotine (CNic; 10~tMnicotine, 20% 02), with or without acetylcholine (ACh) receptor (AChR) agonists/antagonists and blockers of DA uptake. In Nox cultures, extracellular DA, determined by HPLC and normalized to the number of tyrosine hydroxylase-positive glomus cells present, was augmented by acute (-.15-mm) exposure to hypoxia (5% 02;~-~6x basal), high extracellular K~(30 mM; --lOx basal), nomifensine (1 1.sM; a selective DA uptake inhibitor; --.3x basal), and nicotine (100~sM; --5x basal), but not methylcholine (300 1zM; a specific muscarinic agonist). In contrast, in CHox cultures where basal DA release is markedly elevated (--9x control), the stimulatory effect of high K~(3-4x basal) and acute hypoxia (--2x basal) on DA release persisted, but nicotine and nomifensine were no longer effective and methylcholine had a partial inhibitory effect. In CNic cultures, basal DA levels were also elevated (-~9xcontrol), similar to that in CHox cultures; however, although acute hypoxia had a stimulatory effect on DA release (.-2x basal), nicotine, nomifensine, and high K~were ineffective. The elevated basal DA in both CHox and CNic cultures was attenuated by acute or chronic treatment with mecamylamine (100~sfl~, a nicotinic AChR (nAChR) antagonist. In addition, long-term (16-h), but not acute (15-mm), treatment with the muscarinic antagonist atropine (1~M)produced an additional enhancement of basal DA levels in CHox cultures. Thus, after chronic hypoxia or nicotine in vitro, extracellular DA levels around CB chemoreceptor cell clusters appear to be set by a variety of factors including released ACh, positive and negative feedback regulation via nAChRs and muscarinic AChRs, respectively, and modulation of DA transporters. These results provide insight into roles of endogenous transmitters in the adaptation of CB chemoreceptors to chronic hypoxia and suggest pathways by which neuroactive drugs, e.g., nicotine, can interfere with the protective chemoreflex response against hypoxia. Key Words: Dopamine uptake-Glomus cell culture-Nicotinic and muscarinic acetylcholine receptors-a2 chemoreceptors-HPLC.

Carotid body chemoreceptors in dissociated cell culture

“…With respect to the possible interactions between neighboring type 1 cells, recent studies from this laboratory have suggested that several paracrine or autocrine mechanisms are likely to operate, due to presynaptic action of released transmitters. These include both positive and negative feedback effects of ACh (Jackson and Nurse, 1998;Nurse and Zhang, 1999), as well as positive feedback effects of 5-HT . More recent studies on these cultures indicate that the neurotransmitter GABA, acting via GABA B autoreceptors on type 1 cells, is also likely to be an important endogenous modulator of rat carotid body function via a negative feedback pathway ).…”

“…4E), which limits the accumulation of extracellular DA by a re-uptake mechanism. Blockade of this transporter with nomifensine results in a 3-fold increase in extracellular DA levels in rat CB cultures grown under normoxic conditions (Jackson and Nurse, 1998).…”

Signal processing at mammalian carotid body chemoreceptors

“…studies, stimulation of nicotinic receptors activates a non-selective cation current in type I cells, leading to membrane depolarization and an elevation of intracellular Ca 2+ (reviewed in [32]). When applied to whole carotid bodies, nicotine triggers the release of catecholamines [1,32,64], as well as adenosine and ACh [32,49]. However, paracrine actions of ACh are further complicated by the presence of excitatory M1 and inhibitory M2 muscarinic receptors on type I cells in several species [32].…”

“…However, paracrine actions of ACh are further complicated by the presence of excitatory M1 and inhibitory M2 muscarinic receptors on type I cells in several species [32]. There is little doubt that DA is released from type I cells in a Ca 2+ -dependent manner during chemoexcitation [1,23,33,34,64]. With the possible exception of the rabbit carotid body, it is generally agreed that DA is inhibitory, likely acting at pre-and post synaptic sites [1,3,5,6,57].…”

Chronic hypoxia‐induced morphological and neurochemical changes in the carotid body

“…It is reputed to be an excitatory neurotransmitter in the CB (Fitzgerald et al, 1997). Also, acetylcholine may regulate the release of CB DA during CH (Jackson and Nurse, 1998). Recent data suggest that acetylcholine in combination with adenosine triphosphate are coreleased from type I cells during acute hypoxia (Zhang et al, 2000).…”