Hidetada Matsuoka

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.

Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

The up‐regulation of c‐fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c‐fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c‐fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c‐fos gene regulatory sequences that drive the expression of a c‐fos‐eGFP fusion protein. In c‐fos‐eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)‐8, which selectively activates oxytocin (OXT)‐secreting neurones in the hypothalamus. In double transgenic rats that express c‐fos‐eGFP and an OXT‐monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1‐positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK‐8. It is possible that not only a plane image, but also three‐dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

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.

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.

Src mediates endocytosis of TWIK-related acid-sensitive K+ 1 channels in PC12 cells in response to nerve growth factor

TWIK-related acid-sensitive K(+) (TASK) channels produce background K(+) currents. We elucidated that TASK1 channels in rat adrenal medullary cells and PC12 cells are internalized in a clathrin-dependent manner in response to nerve growth factor (NGF). Here, the molecular mechanism for this internalization in PC12 cells was explored. The combination of enzyme inhibitors with tropomyosin receptor kinase A mutants revealed that the internalization was mediated by both phospholipase C and phosphatidylinositol 3-kinase pathways that converge on protein kinase C with the consequent activation of Src, a nonreceptor tyrosine kinase. The NGF-induced endocytosis of TASK1 channels did not occur in the presence of the Src inhibitor or with the expression of a kinase-dead Src mutant. Additionally, NGF induced a transient colocalization of Src with the TASK1 channel, but not the TASK1 mutant, in which tyrosine at 370 was replaced with phenylalanine. This TASK1 mutant showed no increase in tyrosine phosphorylation and markedly diminished internalization in response to NGF. We concluded that NGF induces endocytosis of TASK1 channels via tyrosine phosphorylation in its carboxyl terminus.

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.

GABA Signaling and Neuroactive Steroids in Adrenal Medullary Chromaffin Cells

Gamma-aminobutyric acid (GABA) is produced not only in the brain, but also in endocrine cells by the two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67. In rat adrenal medullary chromaffin cells only GAD67 is expressed, and GABA is stored in large dense core vesicles (LDCVs), but not synaptic-like microvesicles (SLMVs). The α3β2/3γ2 complex represents the majority of GABAA receptors expressed in rat and guinea pig chromaffin cells, whereas PC12 cells, an immortalized rat chromaffin cell line, express the α1 subunit as well as the α3. The expression of α3, but not α1, in PC12 cells is enhanced by glucocorticoid activity, which may be mediated by both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). GABA has two actions mediated by GABAA receptors in chromaffin cells: it induces catecholamine secretion by itself and produces an inhibition of synaptically evoked secretion by a shunt effect. Allopregnanolone, a neuroactive steroid which is secreted from the adrenal cortex, produces a marked facilitation of GABAA receptor channel activity. Since there are no GABAergic nerve fibers in the adrenal medulla, GABA may function as a para/autocrine factor in the chromaffin cells. This function of GABA may be facilitated by expression of the immature isoforms of GAD and GABAA receptors and the lack of expression of plasma membrane GABA transporters (GATs). In this review, we will consider how the para/autocrine function of GABA is achieved, focusing on the structural and molecular mechanisms for GABA signaling.

Molecular mechanism for muscarinic M1 receptor‐mediated endocytosis of TWIK‐related acid‐sensitive K+ 1 channels in rat adrenal medullary cells

The muscarinic acetylcholine receptor (mAChR)‐mediated increase in excitability in rat adrenal medullary cells is at least in part due to inhibition of TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)‐related acid‐sensitive K+ (TASK)1 channels. In this study we focused on the molecular mechanism of mAChR‐mediated inhibition of TASK1 channels. Exposure to muscarine resulted in a clathrin‐dependent endocytosis of TASK1 channels following activation of the muscarinic M1 receptor (M1R). This muscarinic signal for the endocytosis was mediated in sequence by phospholipase C (PLC), protein kinase C (PKC), and then the non‐receptor tyrosine kinase Src with the consequent tyrosine phosphorylation of TASK1. The present results establish that TASK1 channels are tyrosine phosphorylated and internalized in a clathrin‐dependent manner in response to M1R stimulation and this translocation is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells.