Keita Harada

Inhibition of TASK1‐like channels by muscarinic receptor stimulation in rat adrenal medullary cells

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.

Molecular mechanisms supporting a paracrine role of GABA in rat adrenal medullary cells

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 GABAA 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 GABAA 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 GABAA receptors. Increases in Ca2+ 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 Ca2+ 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‐GABAA receptors.

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

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 -60 mV. This inward current was partially suppressed by quinine with an IC(50) of 6.1 μM. The quinine-insensitive component of muscarine-induced currents changed the polarity at -78 mV and was inhibited by bupivacaine, a TWIK-related acid-sensitive K(+) (TASK) channel inhibitor. Conversely, the current-voltage relationship for the bupivacaine-insensitive component of muscarine currents showed a reversal potential of -5 mV and a negative slope below -40 mV. External application of La(3+) 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.

Differential distribution of synaptotagmin-1, -4, -7, and -9 in rat adrenal chromaffin cells

Neurons and certain kinds of endocrine cells, such as adrenal chromaffin cells, have large dense-core vesicles (LDCVs) and synaptic vesicles or synaptic-like microvesicles (SLMVs). These secretory vesicles exhibit differences in Ca2+ sensitivity and contain diverse signaling substances. The present work was undertaken to identify the synaptotagmin (Syt) isoforms present in secretory vesicles. Fractionation analysis of lysates of the bovine adrenal medulla and immunocytochemistry in rat chromaffin cells indicated that Syt 1 was localized in LDCVs and SLMVs, whereas Syt 7 was the predominant isoform present in LDCVs. In contrast to PC12 cells and the pancreatic β cell line INS-1, Syt 9 was not immunodetected in LDCVs in rat chromaffin cells. Double-staining revealed that Syt 9-like immunoreactivity was nearly identical with fluorescent thapsigargin binding, suggesting the presence of Syt 9 in the endoplasmic reticulum (ER).The exogenous expression of Syt 1-GFP in INS-1 cells, which had a negligible level of endogenous Syt 1, resulted in an increase in the amount of Syt 9 in the ER, suggesting that Syt 9 competes with Syt 1 for trafficking from the ER to the Golgi complex. We conclude that LDCVs mainly contain Syt 7, whereas SLMVs contain Syt 1, but not Syt 7, in rat and bovine chromaffin cells.

Storage of GABA in chromaffin granules and not in synaptic-like microvesicles in rat adrenal medullary cells

J. Neurochem. (2010) 114, 617–626.

Localization of Type-2 Angiotensin II Receptor in Adrenal Gland

The localization of the type-2 angiotensin II receptor (AT2) in the adrenal glands of rats, guinea pigs, bovines, and humans was examined at the mRNA and protein levels. PCR products for AT2 were detected in the adrenal cortices and adrenal medullae of all the mammals examined with an RT-PCR technique. Three different anti-AT2 antibodies (Abs), whose specificity was confirmed in our hands, recognized a 50-kDa protein in the adrenal glands of the four mammals, and this recognition was abolished by the preabsorption of an Ab with an antigen. Immunoblotting and immunohistochemistry revealed that the 50-kDa protein was expressed consistently and variably in the adrenal cortices and medullae of various mammals, respectively. We conclude that the 50-kDa AT2 is consistently expressed in the adrenal cortex in a wide variety of mammals.

Paracrine Role of GABA in Adrenal Chromaffin Cells

The function of GABA in the adrenal medulla is still controversial. We will review experimental results in vivo and in vitro in adrenal chromaffin cells of various mammals to clarify what has been elucidated and what still remains to be settled.

Identification of muscarinic receptor subtypes involved in catecholamine secretion in adrenal medullary chromaffin cells by genetic deletion

Activation of muscarinic receptors results in catecholamine secretion in adrenal chromaffin cells in many mammals, and muscarinic receptors partly mediate synaptic transmission from the splanchnic nerve, at least in guinea pigs. To elucidate the physiological functions of muscarinic receptors in chromaffin cells, it is necessary to identify the muscarinic receptor subtypes involved in excitation.

Ca2+ pathway involved in the refilling of store sites in rat adrenal medullary cells

It has been suggested that store-operated Ca(2+) entry (SOC) facilitates catecholamine secretion and synthesis in bovine adrenal medullary (AM) cells. However, there has been no experimental result clearly showing that cation channel activity is enhanced by store Ca(2+) depletion. Thus the present experiments were undertaken to address the issue of whether rat AM cells have SOC channels. Inhibition of the sarco(endo)plasmic reticulum Ca(2+) (SERCA) pump resulted in a sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat AM cells. This increase was completely suppressed by 2 mM Ni(2+) but not by 100 muM D600. A bath application of Ni(2+), but not D600, produced an outward current at -60 mV in rat AM cells, whereas exposure to a SERCA pump inhibitor did not affect either the whole cell current level or the Ni(2+)-induced outward current. The refilling of intracellular store sites was suppressed by the addition of Ni(2+) to the perfusate. RT-PCR revealed that transcripts for transient receptor potential channels 1 (TRPC1) and 5 (TRPC5) were present in rat adrenal medullas. Immunocytochemistry showed that TRPC1 channels, which have been implicated in SOC in certain types of cells, were mainly localized in the endoplasmic reticulum (ER) and not in the plasma membrane, and that STIM1, a Ca(2+) sensor in the ER, was not expressed in rat AM cells. On the basis of these results, we conclude that rat AM cells lack the SOC mechanism.

Muscarinic receptors in adrenal chromaffin cells: physiological role and regulation of ion channels

Adrenal medullary chromaffin cells in mammals are innervated by sympathetic preganglionic nerve fibers, as are sympathetic ganglion neurons. Acetylcholine in the ganglion neurons is well established as mediating fast and slow excitatory postsynaptic potentials through nicotinic and muscarinic acetylcholine receptors (AChRs), respectively. The role of muscarinic AChRs during neuronal transmission in chromaffin cells varies among different mammals. Furthermore, the ion channel mechanisms associated with the muscarinic AChR-mediated increase in excitability of chromaffin cells are complicated and different from the excitation of ganglion neurons, which has been ascribed to the inhibition of M-type K+ channels. In this review, we focus on muscarinic receptor-mediated excitation in rodent and guinea pig chromaffin cells, in particular, on the role of muscarinic receptors in neuronal transmission, the muscarinic receptor subtypes involved in excitation and secretion, and the muscarinic regulation of ion channels including TWIK-related acid-sensitive K+ channels. Finally, we discuss prospectively the future of muscarinic receptor research in adrenal chromaffin cells.