Identification and Role of Muscarinic Receptor Subtypes Expressed in Rat Adrenal Medullary Cells

Abstract

Abstract. The muscarinic receptor is known to be involved in the acetylcholine (ACh)-induced secretion of catecholamines in the adrenal medullary (AM) cells of various mammals. The muscarinic receptor subtype involved and its physiological role, however, have not been elucidated yet. Thus, we investigated these issues in acutely isolated rat AM cells and perfused rat adrenal medulla. The RT-PCR analysis revealed the presence of M 2 , M 3 , M 4 , and M 5 mRNAs. Immunocytochemistry with specific antibodies showed that M 5 -like immunoreactivities (IRs) were detected at half the cell membrane area, which was much larger than that with M 3 -or M 4 -like IRs. Muscarine produced inward currents in a dose-dependent manner. Pilocarpine, McN-A-343, and oxotremorine were less efficient than muscarine; and RS-86, which has no action on the M 5 receptor, produced no current. Electrical stimulation of nerve fibers produced a frequency-dependent increase in the Ca 2+ signal in perfused adrenal medullae. Muscarinic receptors were found to be involved in neuronal transmission in AM cells in the presence of a cholinesterase inhibitor, which suppresses ACh degradation. We concluded that the M 5 receptor is the major muscarinic receptor subtype in rat AM cells and may be involved in neuronal transmission under conditions where ACh spills over the synapse.

Mechanisms and roles of muscarinic activation in guinea-pig adrenal medullary cells

“…Oligo dT primer was utilized for the RT reaction to obtain cDNAs. PCR reactions were carried out with 1.25 l of DNA template, 4 pmol of primers (20), 2 mM of dNTPs, 0.5 U of rTaq (Takara, Otsu, Japan), and PCR buffers supplied with the kit in a final volume of 20 l. The PCR protocol used started with an initial 3-min denaturation step at 94°C, followed by 30 to 40 cycles of the profile consisting of 30 s of denaturation at 94°C, 30 s of annealing at 54°C to 60°C, and 30 s of extension at 72°C. To obtain the maximum fidelity, a hot-start procedure was used.…”

“…Muscarinic receptors, however, have biochemically and/or functionally been detected in guinea-pig (22,26), cat (2), bovine (2,14), porcine (17), chicken (32), rat (23,38,39), and human (43) AM cells. In rat AM cells, stimulation of the M5 muscarinic receptors has been suggested to induce inhibition of TWIK-related acid-sensitive K ϩ channel 1 (TASK1) activity with the consequent secretion of catecholamine (20,23). This muscarinic signaling was involved in synaptic transmission from the preganglionic nerve fiber only under conditions where the degradation of ACh by cholinesterase was impaired (20).…”

“…In rat AM cells, stimulation of the M5 muscarinic receptors has been suggested to induce inhibition of TWIK-related acid-sensitive K ϩ channel 1 (TASK1) activity with the consequent secretion of catecholamine (20,23). This muscarinic signaling was involved in synaptic transmission from the preganglionic nerve fiber only under conditions where the degradation of ACh by cholinesterase was impaired (20). In contrast, catecholamine secretion evoked by electrical stimulation of nerve fibers was partially suppressed by application of atropine, a muscarinic antagonist, in isolated perfused adrenal glands of spontaneously hypertensive rats (SHRs) but not normotensive Wistar-Kyoto (WKY) rats (39), suggesting plasticity of the muscarinic signaling in AM cells.…”

Ca2+ Imaging in Perfused Adrenal Medullae

“…Administration of nicotine or GABA resulted in an increase in fluorescence intensity, which decayed due to desensitization of receptors in the presence of the agonist. The Ca 2+ signal in response to electrical stimulation during application of nicotine and GABA also diminished markedly, probably because of occlusion of nicotinic ACh receptors with nicotine [13] and a shunt effect by activation of GABA A receptors [11], respectively. However, saturation of Ca 2+ signal can not be excluded.…”

“…To accomplish this goal, we have developed a special technique to obtain Ca 2+ image from adrenal medulla which are perfused retrogradely through the adrenal vein [10]. With this method, the properties of Ca 2+ store sites [10], the effects of γ-aminobutyric acid (GABA) on neuronally evoked increase in excitability [11,12], and the role of muscarinic receptors in neurotransmission in rat AM cells [13] have been investigated in the past decade. Because of the technical difficulty, however, this method is not common.…”