RESUMO
Adrenal medullary chromaffin (AMC) cells in the perinatal period and carotid body glomus cells after birth respond to hypoxia with catecholamine secretion. The hypoxia detection mechanism in such O2-sensitive cells is still not well defined. One hypothesis is that a decrease in cellular ATP may be involved in the hypoxia detection. This idea is based on ATP dependence of TASK channel activity that regulates the resting membrane potential and is suppressed by hypoxia in glomus cells. Mitochondrial ATPase inhibitor factor-1 (IF1), a physiological regulator of ATP synthase, helps prevent ATP hydrolysis under hypoxic conditions. In cells where IF1 expression is high, exposure to hypoxia is expected to have no effect on TASK channel activity. This possibility was electrophysiologically and immunocytochemically explored. Single channel recordings revealed that 36-pS TASK3-like channels contribute to the resting membrane potential in young rat adrenal cortical (AC) cells. TASK3-like channel activity in a cell-attached patch was not affected by bath application of mitochondrial inhibitors. Consistent with this finding, IF1-like immunoreactive material was well expressed in rat AC cells. In further support of our hypothesis, IF1-like immunoreactive material was well expressed in adult rat AMC cells that are known to be hypoxia-insensitive and minimally expressed in newborn AMC cells that are hypoxia-sensitive. These results provide evidence for the functional relevance of IF1 expression in excitability in O2-sensitive cells in response to mitochondrial inhibition.
Assuntos
Células Cromafins , Gravidez , Feminino , Ratos , Animais , Hipóxia Celular , Células Cromafins/metabolismo , Mitocôndrias/metabolismo , Hipóxia/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Chronic intermittent hypoxia (CIH) has been used as a model to mimic nocturnal apnea, which is associated with hypertension. One of the mechanisms for hypertension in patients with nocturnal apnea is an enhancement of the plasma membrane response to acute hypoxia in carotid body glomus cells. Hypoxia is known to induce depolarization via inhibiting TWIK-related acid-sensitive K+ (TASK) channels, one type of leak K+ channels, in glomus cells. The present experiment was undertaken to immunocytochemically investigate the effects of CIH on the expression and intracellular localization of TASK1 channels and p11 that critically affect the trafficking of TASK1 to the cell surface. The expression levels of TASK1 proteins and p11 and their intracellular localization in rat carotid body glomus cells were not noticeably affected by CIH, suggesting that the enhanced membrane response to acute hypoxia is not due to an increase in surface TASK channels.
Assuntos
Corpo Carotídeo , Hipertensão , Animais , Apneia/metabolismo , Corpo Carotídeo/metabolismo , Hipóxia/metabolismo , RatosRESUMO
γ-Aminobutyric acid (GABA) is thought to play a paracrine role in adrenal medullary chromaffin (AMC) cells. Comparative physiological and immunocytochemical approaches were used to address the issue of how the paracrine function of GABA in AMC cells is established. GABAA receptor Cl- channel activities in AMC cells of rats and mice, where corticosterone is the major glucocorticoid, were much smaller than those in AMC cells of guinea-pigs and cattle, where cortisol is the major. The extent of enhancement of GABAA receptor α3 subunit expression in rat pheochromocytoma (PC12) cells by cortisol was larger than that by corticosterone in parallel with their glucocorticoid activities. Thus, the species difference in GABAA receptor expression may be ascribed to a difference in glucocorticoid activity between corticosterone and cortisol. GABAA receptor Cl- channel activity in mouse AMC cells was enhanced by allopregnanolone, as noted with that in guinea-pig AMC cells, and the enzymes involved in allopregnanolone production were immunohistochemically detected in the zona fasciculata in both mice and guinea pigs. The expression of glutamic acid decarboxylase 67 (GAD67), one of the GABA synthesizing enzymes, increased after birth, whereas GABAA receptors already developed at birth. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, but not nicotinic or muscarinic receptors, in PC12 cells, resulted in an increase in GAD67 expression in a protein-kinase A-dependent manner. The results indicate that glucocorticoid and PACAP are mainly responsible for the expressions of GABAA receptors and GAD67 involved in GABA signaling in AMC cells, respectively.
Assuntos
Medula Suprarrenal/fisiologia , Células Cromafins/fisiologia , Comunicação Parácrina/fisiologia , Ácido gama-Aminobutírico/fisiologia , Medula Suprarrenal/citologia , Animais , Bovinos , Canais de Cloreto/metabolismo , Cricetinae , Glutamato Descarboxilase/metabolismo , Cobaias , Hidrocortisona/metabolismo , Imuno-Histoquímica , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Células PC12 , Pregnanolona/farmacologia , Ratos , Receptores de GABA-A/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/efeitos dos fármacos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismoRESUMO
Muscarinic receptor stimulation results in activation of nonselective cation (NSC) channels in guinea pig adrenal medullary (AM) cells. The biophysical and pharmacological properties of the NSC channel suggest the involvement of heteromeric channels of TRPC1 with TRPC4 or TRPC5. This possibility was explored in PC12 cells and guinea pig AM cells. Proximity ligation assay (PLA) revealed that when exogenously expressed in PC12 cells, TRPC1 forms a heteromeric channel with TRPC4, but not with TRPC5, in a STIM1-dependent manner. The heteromeric TRPC1-TRPC4 channel was also observed in AM cells and trafficked to the cell periphery in response to muscarine stimulation. To explore whether heteromeric channels are inserted into the cell membrane, tags were attached to the extracellular domains of TRPC1 and TRPC4. PLA products developed between the tags in cells stimulated by muscarine, but not in resting cells, indicating that muscarinic stimulation results in the membrane insertion of channels. This membrane insertion required expression of full-length STIM1. We conclude that muscarinic receptor stimulation results in the insertion of heteromeric TRPC1-TRPC4 channels into the cell membrane in PC12 cells and guinea pig AM cells.
Assuntos
Membrana Celular/metabolismo , Receptores Muscarínicos/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Canais de Cátion TRPC/metabolismo , Medula Suprarrenal/citologia , Medula Suprarrenal/metabolismo , Animais , Linhagem Celular , Cobaias , Masculino , Células PC12 , Domínios Proteicos , RatosRESUMO
TWIK-related acid-sensitive K+ (TASK) channels contribute to the resting membrane potential in various kinds of cells, such as brain neurons, smooth muscle cells, and endocrine cells. Loss-of-function mutations at multiple sites in the KCNK3 gene encoding for TASK1 channels are one of the causes of pulmonary arterial hypertension in humans, whereas a mutation at only one site is reported for TASK3 channels, resulting in a syndrome of mental retardation, hypotonia, and facial dysmorphism. TASK channels are subject to regulation by G protein-coupled receptors (GPCRs). Two mechanisms have been proposed for the GPCR-mediated inhibition of TASK channels: a change in gating and channel endocytosis. The most feasible mechanism for altered gating is diacylglycerol binding to a site in the C-terminus, which is shared by TASK1 and TASK3. The inhibition of channel function by endocytosis requires the presence of a tyrosine residue subjected to phosphorylation by the non-receptor tyrosine kinase Src and a dileucine motif in the C-terminus of TASK1. Therefore, homomeric TASK1 and heteromeric TASK1-TASK3 channels, but not homomeric TASK3, are internalized by GPCR stimulation. Tyrosine phosphorylation by Src is expected to result in a conformational change in the C-terminus, allowing for AP-2, an adaptor protein for clathrin, to bind to the dileucine motif. It is likely that a raft membrane domain is a platform where TASK1 is located and the signaling molecules protein kinase C, Pyk2, and Src are recruited in sequence in response to GPCR stimulation.
Assuntos
Canalopatias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Transporte Proteico/fisiologia , Animais , Humanos , Fosforilação/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologiaRESUMO
External acidity induces catecholamine secretion by inhibiting TASK1-like channels in rat adrenal medullary (AM) cells. TASK channels can function as a heteromer or homomer in the TASK subfamily. In this study, we elucidate the molecular identity of TASK1-like channels in mouse AM cells using gene knockout. Genetic deletion of TASK1, but not TASK3, abolished the depolarizing inward current and catecholamine secretion in response to acidity, whereas it did not affect the resting current level. Immunocytochemistry revealed that AM cells exhibited predominantly TASK1-like and little TASK3-like immunoreactivity. A proximity ligation assay showed that TASK1/3 heteromeric channels were not formed in AM cells or PC12 cells. However, the exogenous expression of p11 in PC12 cells resulted in the heteromeric formation of TASK isoforms, which were mainly located in the cytoplasm, and p11 was not expressed in rat adrenal medullae or PC12 cells. In AM cells, genetic deletion of TASK1 resulted in enhancement of the immunoreactivity of the TALK2 channel, but not TASK3. The results indicate that TASK1 homomeric channels function as acidity sensors in AM cells, and that function is facilitated by the lack of p11 expression.-Inoue, M., Matsuoka, H., Lesage, F., Harada, K. Lack of p11 expression facilitates acidity-sensing function of TASK1 channels in mouse adrenal medullary cells.
Assuntos
Canais Iônicos Sensíveis a Ácido/fisiologia , Ácidos/química , Medula Suprarrenal/fisiologia , Anexina A2/deficiência , Proteínas do Tecido Nervoso/fisiologia , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Canais de Potássio/fisiologia , Proteínas S100/deficiência , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células PC12 , RatosRESUMO
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.
Assuntos
Medula Suprarrenal/citologia , Células Cromafins/metabolismo , Canais de Potássio/metabolismo , Receptores Muscarínicos/metabolismo , Canais de Cátion TRPC/metabolismo , Potenciais de Ação , Medula Suprarrenal/metabolismo , Animais , Células Cromafins/fisiologia , Humanos , Receptores Muscarínicos/genéticaRESUMO
M-type K+ channels contribute to the resting membrane potential in the sympathetic ganglion neurons of various animals, whereas their expression in adrenal medullary (AM) cells has been controversial. The present experiment aims to explore the expression of M channels comprising the KCNQ2 subunit in the rat AM cell and its immortalized cell line PC12 cells at the protein level and how its expression in PC12 cells is regulated. The KCNQ2 isoform was recognized in homogenates of PC12 cells but not the rat adrenal medullae by immunoblotting and KCNQ2-like immunoreactivity (IR) was detected in PC12 cells but not in rat AM cells. When the PC12 cells were maintained in a dexamethasone-containing medium, KCNQ2-like IR in the cells was suppressed, whereas the removal of fetal bovine serum from the culture medium for 1 day resulted in an increase in KCNQ2-like IR. A similar enhancement occurred when PC12 cells were cultured under conditions where glucocorticoid receptor (GR) and/or mineralocorticoid receptor (MR) activities were suppressed. These morphological findings were confirmed in functional analysis. The cells cultured in the presence of an inhibitor of either GR or MR exhibited larger amplitudes of Ca2+ signal in response to an M channel inhibitor than did the cells in its absence, whereas the resting Ca2+ level in the former was lower than that in the latter. These results indicate that the M channel is not expressed in rat AM cells and this absence of expression may be ascribed to the suppression by glucocorticoid activity.
Assuntos
Medula Suprarrenal/citologia , Medula Suprarrenal/metabolismo , Canal de Potássio KCNQ2/metabolismo , Animais , Glucocorticoides/sangue , Proteínas de Fluorescência Verde/metabolismo , Masculino , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Células PC12 , Ratos , Ratos Wistar , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismoRESUMO
The published online version contains some mistakes for the additional corrections were missed by typesetter which also include the replacement of Fig. 5.
RESUMO
KEY POINTS: 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 (M1 R). 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 M1 R stimulation and this translocation is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells. ABSTRACT: Activation of muscarinic receptor (mAChR) in rat adrenal medullary (AM) cells induces depolarization through the inhibition of TWIK-related acid-sensitive K+ (TASK)1 channels. Here, pharmacological and immunological approaches were used to elucidate the molecular mechanism for this mAChR-mediated inhibition. TASK1-like immunoreactive (IR) material was mainly located at the cell periphery in dissociated rat AM cells, and its majority was internalized in response to muscarine. The muscarine-induced inward current and translocation of TASK1 were suppressed by dynasore, a dynamin inhibitor. The muscarinic translocation was suppressed by MT7, a specific M1 antagonist, and the dose-response curves for muscarinic agonist-induced translocation were similar to those for the muscarinic inhibition of TASK1 currents. The muscarine-induced inward current and/or translocation of TASK1 were suppressed by inhibitors for phospholipase C (PLC), protein kinase C (PKC), and/or Src. TASK1 channels in AM cells and PC12 cells were transiently associated with Src and were tyrosine phosphorylated in response to muscarinic stimulation. After internalization, TASK1 channels were quickly dephosphorylated even while they remained in the cytoplasm. The cytoplasmic TASK1-like IR material quickly recycled back to the cell periphery after muscarine stimulation for 0.5 min, but not 10 min. We conclude that M1 R stimulation results in internalization of TASK1 channels through the PLC-PKC-Src pathway with the consequent phosphorylation of tyrosine and that this M1 R-mediated internalization is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells.
Assuntos
Medula Suprarrenal/citologia , Endocitose , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Receptores Muscarínicos/metabolismo , Medula Suprarrenal/metabolismo , Animais , Células Cultivadas , Masculino , Proteínas do Tecido Nervoso , Células PC12 , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Fosfolipases Tipo C/metabolismo , Quinases da Família src/metabolismoRESUMO
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.
Assuntos
Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Fator de Crescimento Neural/farmacologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Quinases da Família src/fisiologia , Animais , Masculino , Proteínas do Tecido Nervoso , Células PC12 , RatosRESUMO
TWIK-related acid-sensitive K(+) (TASK) channels belong to a family of two-pore domain K(+) channels which produce background K(+) currents and are involved in important physiological functions, such as acidosis detection. We have recently elucidated that TASK1-like channels function as a sensor of acidosis in rat adrenal medullary (AM) cells and thus are indispensable for the endocrine function of AM cells. Here, using pharmacological, electrophysiological and biochemical methods, we studied how the expression and localisation of TASK1 channels are regulated in rat AM cells and PC12 cells. PC12 cells were found to express not only TASK1 but also TASK3 channels, and they did not constitute a heterodimer. The exposure of AM cells and PC12 cells to nerve growth factor (NGF) induced endocytosis of TASK1, but not TASK3 channels, in a clathrin-dependent manner. Mutation analysis of the TASK1 channel revealed that the dileucine motif (LL263/264) was involved in at least part of the endocytosis. Plating GFP-TASK1-expressing PC12 cells onto a sheet of fibroblasts, which produced NGF, resulted in the endocytosis of GFP-TASK1 channels. Additionally, the expression of TASK1 channels at the protein and mRNA levels was suppressed in PC12 cells treated with NGF for 2 weeks. These results indicate that NGF suppresses the expression of TASK1 channels in the plasma membrane via not only endocytosis but also the inhibition of gene transcription. Thus, no access to NGF may play a major role for the maintenance of TASK1 channels in the cell membrane in AM cells.
Assuntos
Medula Suprarrenal/metabolismo , Endocitose/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Medula Suprarrenal/citologia , Motivos de Aminoácidos , Animais , Vesículas Revestidas por Clatrina/metabolismo , Células Endócrinas/efeitos dos fármacos , Células Endócrinas/metabolismo , Masculino , Mutação , Proteínas do Tecido Nervoso , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/genética , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Transcrição Gênica/genéticaRESUMO
One of the mechanisms for hypertension is an increase in blood catecholamines due to increased secretion from sympathetic nerve terminals and adrenal medullary chromaffin (AMC) cells. Spontaneously hypertensive rats (SHRs) are used as an animal model of hypertension. Catecholamine secretion in AMC cells occurs in response to humoral factors and neuronal inputs from the sympathetic nerve fibres. Acetylcholine (ACh) released from the nerve terminals activates nicotinic as well as muscarinic ACh receptors. The present experiment aimed to elucidate whether muscarinic receptor-mediated excitation is altered in SHR AMC cells and, if it is, how. Compared with normotensive rat AMC cells, muscarinic stimulation induced greater catecholamine secretion and larger depolarising inward currents in SHR AMC cells. In contrast to normotensive rat AMC cells, the muscarine-induced current consisted of quinine-sensitive and quinine-insensitive components. The former and the latter are possibly ascribed to nonselective cation channel activation and TWIK-related acid-sensitive K+ (TASK) channel inhibition, as noted in guinea pig AMC cells. In fact, immunoreactive material for TASK1 and several isoforms of transient receptor potential canonical (TRPC) channels was detected in SHR AMC cells. Stromal interaction molecule 1 (STIM1), which plays an essential role for heteromeric TRPC1-TRPC4 channel formation and is not expressed in normotensive rat AMC cells, was detected in the cytoplasm and co-localised with TRPC1. The expression of muscarinic M1 receptors was enhanced in SHR AMC cells compared with normotensive rats. The results indicate that muscarinic excitation is enhanced in SHR AMC cells, probably through facilitation of TRPC channel signalling.
Assuntos
Medula Suprarrenal , Células Cromafins , Hipertensão , Ratos , Animais , Cobaias , Ratos Endogâmicos SHR , Quinina/metabolismo , Células Cromafins/metabolismo , Medula Suprarrenal/metabolismo , Receptores Muscarínicos/metabolismo , Colinérgicos/metabolismo , Hipertensão/metabolismo , Catecolaminas/metabolismoRESUMO
Adrenal medullary chromaffin (AMC) and sympathetic ganglion cells are derived from the neural crest and show a similar developmental path. Thus, these two cell types have many common properties in membrane excitability and signaling. However, AMC cells function as endocrine cells while sympathetic ganglion cells are neurons. In rat sympathetic ganglion cells, muscarinic M1 and M4 receptors mediate excitation and inhibition via suppression of M-type K+ channels and suppression of voltage-dependent Ca2+ channels, respectively. On the other hand, M1 receptor stimulation in rat AMC cells also produces excitation by suppressing TWIK-related acid sensitive K+ (TASK) channels. However, whether M4 receptors are coupled with voltage-dependent Ca2+ channel suppression is unclear. We explore this issue electrophysiologically and biochemically. Electrical stimulation of nerve fibers in rat adrenal glands trans-synaptically increased the Ca2+ signal in AMC cells. This electrically evoked increased Ca2+ signal was not altered during muscarine-induced increase in Ca2+ signal, whereas it decreased significantly during a GABA-induced increase, due to a shunt effect of increased Cl- conductance. The whole-cell current recordings revealed that voltage-dependent Ca2+ currents in AMC cells were suppressed by adenosine triphosphate, but not by muscarinic agonists. The fractionation analysis and immunocytochemistry indicated that CaV1.2 Ca2+ channels and M4 receptors are located in the raft and non-raft membrane domains, respectively. We concluded that muscarinic stimulation in rat AMC cells does not produce voltage-dependent Ca2+ channel inhibition. This lack of muscarinic inhibition is at least partly due to physical separation of voltage-dependent Ca2+ channels and M4 receptors in the plasma membrane.
RESUMO
Immunocytochemistry enables the detection and localization of proteins in cells that are acutely dissociated or in culture. There are advantages and disadvantages to the use of cultured cells for immunocytochemistry. One of the advantages is that cultured cells can be used for one or more weeks after the dissociation of cells, whereas one of the disadvantages is that the properties of cells in culture might change under artificial conditions. On the other hand, acutely dissociated cells are expected to have the original properties of cells because almost all procedures before fixation, except for enzymatic digestion, are carried out at low temperatures. Here, we describe how adrenal medullary cells of small animals are acutely dissociated for immunostaining.
Assuntos
Medula Suprarrenal , Células Cromafins , Células Endócrinas , Animais , Células Cultivadas , Imuno-HistoquímicaRESUMO
This study aimed to develop a cementitious repair material that can be constructed in cold weather conditions. The addition of nitrite/nitrate-based antifreezing agents has been shown to increase the initial strength of cementitious repair materials in cold weather. However, increasing the amount of these agents may lead to an increase in deformation behavior and shrinkage cracking. In this study, the effects of different types and amounts of nitrite/nitrate-based antifreezing agents on the strength development and deformation behavior of cementitious repair materials under low-temperature conditions were evaluated. As a result, it was found that the addition of a large amount of calcium nitrite can promote hydration and improve the initial strength of the repair material, irrespective of the type of antifreezing agent. However, this also leads to an increase in shrinkage and the concern of shrinkage cracking. Therefore, a repair material that is repairable in winter was developed by balancing the initial strength and deformation behavior through the appropriate selection of antifreezing agents. The developed repair material can be used to repair structures in cold weather conditions, which is of great significance for the construction industry in Hokkaido, Japan.
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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.
Assuntos
Medula Suprarrenal/citologia , Medula Suprarrenal/metabolismo , Receptores Muscarínicos/fisiologia , Medula Suprarrenal/efeitos dos fármacos , Animais , Permeabilidade da Membrana Celular/fisiologia , Cobaias , Masculino , Muscarina/farmacologia , Proteínas do Tecido Nervoso/fisiologia , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Ratos , Receptores Muscarínicos/metabolismoRESUMO
The Ca2+ imaging method was developed to explore changes in excitability in adrenal medullary (AM) cells in a large field in response to synaptic input and chemicals. The adrenal medullae of rats and guinea pigs were retrogradely loaded with Ca2+ indicator through the adrenal vein. Nerve fibers remaining in the adrenal gland were electrically stimulated to induce postsynaptic responses in AM cells, and chemicals were applied to the cells by adding to the perfusate. With this method, gamma-aminobutyric acid (GABA) was shown to increase the Ca2+ signal in almost all and 40% AM cells in guinea pigs and rats, respectively.
Assuntos
Medula Suprarrenal/inervação , Cálcio , Medula Suprarrenal/efeitos dos fármacos , Animais , Estimulação Elétrica , Cobaias , Masculino , Perfusão , Ratos , Ratos Wistar , Ácido gama-Aminobutírico/farmacologiaRESUMO
TWIK-related acid-sensitive K+ (TASK) channels are thought to contribute to the resting membrane potential in adrenal cortical (AC) cells. However, the molecular identity of TASK channels in AC cells have not yet been elucidated. Thus, immunocytochemical and molecular biological approaches were employed to investigate the expression and intracellular distribution of TASK1 and TASK3 in mouse AC cells and H295R cells derived from human adrenocortical carcinoma. Immunocytochemical study revealed that immunoreactive materials were mainly located in the cytoplasm for TASK1 and at the cell periphery for TASK3 in mouse AC cells. A similar pattern of localization was observed when GFP-TASK1 and GFP-TASK3 were exogenously expressed in H295R cells. In addition, p11 that is known to suppress the endoplasmic reticulum exit of TASK1 was localized in the cytoplasm in mouse AC and H295R cells, but not in adrenal medullary cells. Proximity ligation assay (PLA) suggested formation of heteromeric TASK1-3 channels that were found predominantly in the cytoplasm and weakly at the cell periphery. A similar distribution was observed following exogenous expression of tandem TASK1-3 channels in H295R cells. When stimulated by angiotensin II, however, tandem TASK1-3 channels were present mainly in the cytoplasm in all H295R cells. In contrast to that in H295R cells, tandem channels were exclusively located at the cell periphery in all non-stimulated and exclusively in the cytoplasm in stimulated PC12 cells, respectively. From these results, we conclude that TASK1 proteins are present mainly in the cytoplasm and minimally at the cell periphery as a heteromeric channel with TASK3, whereas the majority of TASK3 is at the cell periphery as homomeric and heteromeric channels.
Assuntos
Células Endócrinas , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Angiotensina II/metabolismo , Animais , Células Endócrinas/metabolismo , Humanos , Camundongos , Células PC12 , Canais de Potássio/metabolismo , RatosRESUMO
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 Ca(2+) 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 ß 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.