The muscarinic receptor is known to be involved in the acetylcholine‐induced secretion of catecholamines in the adrenal medulla (AM) cells of various mammals. The ionic mechanisms, however, have not been elucidated yet. Thus, we investigated the issue in acutely isolated rat AM cells with the perforated patch clamp method. Bath application of 30 μM muscarine induced depolarization with the consequent generation of action potentials or an inward current at negative membrane potentials. The muscarine‐sensitive current instantaneously changed in amplitude upon application of command pulses without a time‐dependent component, altered the polarity as a K+‐electrode, and showed rectification of the Goldman‐Hodgkin‐Katz (GHK) type. The whole‐cell current at −20 mV was inhibited by external H+ ions with a concentration responsible for half inhibition of pH 7.09 and muscarine failed to induce a further inward current during exposure to a saline in which pH decreased to 6.5. A similar occlusion occurred in secretion when pH in muscarine‐containing saline decreased to 6.6. RT‐PCR, immunoblotting, and immunocytochemistry suggested that rat AM cells mainly express the TASK1 channel. This TASK channel in AM cells may directly sense a decrease in blood pH, which occurs during exercise. The muscarine action was mimicked by oxotremorine–methiodide, but not by oxotremorine. The present results indicate that activation of muscarinic receptors or a decrease in external pH in the rat AM cell induces secretion through the inhibition of TASK1‐like channels.

GABA is known to produce membrane depolarization and secretion in adrenal medullary (AM) cells in various species. However, whether the GABAergic system is intrinsic or extrinsic or both in the adrenal medulla and the role that GABA plays are controversial. Therefore, these issues were addressed by combining a biochemical and functional analysis. Glutamic acid decarboxylase (GAD), a GABA synthesizing enzyme, and vesicular GABA transporter (VGAT) were expressed in rat AM cells at the mRNA and protein levels, and the adrenal medulla had no nerve fibre-like structures immunoreactive to an anti-GAD Ab. The double staining for VGAT and chromogranin A indicates that GABA was stored in chromaffin granules. The α1, α3, β2/3, γ2 and δ subunits of GABA A receptors were identified in AM cells at the mRNA and protein levels. Pharmacological properties of GABA-induced Cl − currents, immunoprecipitation experiments and immunocytochemistry indicated the expression of not only γ2-, but also δ-containing GABA A receptors, which have higher affinities for GABA and neurosteroids. Expression of GATs, which are involved in the clearance of GABA at GABAergic synapses, were conspicuously suppressed in the adrenal medulla, compared with expression levels of GABA A receptors. Increases in Ca 2+ signal in AM cells evoked trans-synaptically by nerve stimulation were suppressed during the response to GABA, and this suppression was attributed to the shunt effect of the GABA-induced increase in conductance. Overall Ca 2+ responses to electrical stimulation and GABA in AM cells were larger or smaller than those to electrical stimulation alone, depending on the frequency of stimulation. The results indicate that GABA functions as a paracrine in rat AM cells and this function may be supported by the suppression of GAT expression and the expression of not only γ2-, but also δ-GABA A receptors.

— With a view toward elucidating the physiocochemical properties of the nerve membrane, the behavior of various dye molecules incorporated into the membrane was investigated. By measuring the difference between the absorption spectra of dyes in the membrane at rest and those during nerve excitation, the nature of the conformation changes of the membrane macromolecules were examined. Mathematical expressions were developed describing the absorbance changes of dye molecules during nerve excitation. Experimental evidence was presented indicating that there is, during nerve excitation, rotation of dye molecules in the membrane. The physicochemical bases of the process of production of fluorescence changes during nerve excitation are discussed. Based on the results of measurements of the polarization of the fluorescent light, the existence of a highly ordered macromolecular structure at or near the inner surface of the membrane was inferred.

The effect of sea anemone toxins from Parasicyonis actinostoloides and Anemonia sulcata on the Na conductance in crayfish giant axons was studied under voltage-clamp conditions . The toxin slowed the Na inactivation process without changing the kinetics of Na activation or K activation in an early stage of the toxin effect . An analysis of the Na current profile during the toxin treatment suggested an all-or-none modification of individual Na channels . Toxin-modified Na channels were partially inactivated with a slower time course than that of the normal inactivation . This slow inactivation in steady state decreased in its extent as the membrane was depolarized to above -45 mV, so that practically no inactivation occurred at the membrane potentials as high as +50 mV . In addition to inhibition of the normal Na inactivation, prolonged toxin treatment induced an anomalous closing in a certain population of Na channels, indicated by very slow components of the Na tail current . The observed kinetic natures of toxin-modified Na channels were interpreted based on a simple scheme which comprised interconversions between functional states of Na channels . The voltage dependence of Parasicyonis toxin action, in which depolarization caused a suppression in development of the toxin effect, was also investigated .

Muscarinic receptors are expressed in the adrenal medullary (AM) cells of various mammals, but their physiological roles are controversial. Therefore, the ionic mechanism for muscarinic receptor-mediated depolarization and the role of muscarinic receptors in neuronal transmission were investigated in dissociated guinea-pig AM cells and in the perfused guinea-pig adrenal gland. Bath application of muscarine induced an inward current at Ϫ60 mV. This inward current was partially suppressed by quinine with an IC50 of 6.1 M. The quinineinsensitive component of muscarine-induced currents changed the polarity at Ϫ78 mV and was inhibited by bupivacaine, a TWIKrelated acid-sensitive K ϩ (TASK) channel inhibitor. Conversely, the current-voltage relationship for the bupivacaine-insensitive component of muscarine currents showed a reversal potential of Ϫ5 mV and a negative slope below Ϫ40 mV. External application of La 3ϩ had a double action on muscarine currents of both enhancement and suppression. Immunoblotting and immunocytochemistry revealed expression of TASK1 channels and cononical transient receptor potential channels 1, 4, 5, and 7 in guinea-pig AM cells. Retrograde application of atropine reversibly suppressed transsynaptically evoked catecholamine secretion from the adrenal gland. The results indicate that muscarinic receptor stimulation in guinea-pig AM cells induces depolarization through inhibition of TASK channels and activation of nonselective cation channels and that muscarinic receptors are involved in neuronal transmission from the splanchnic nerve.

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.

No abstract

No abstract