DHEA attenuates catecholamine secretion from bovine adrenal chromaffin cells

Dehydroepiandrosterone (DHEA) is a putative anti-stress agent and stress is associated with the secretion of catecholamine from the adrenal gland, but the effects of DHEA on catecholamine secretion are not fully understood. Using bovine chromaffin cells, we found that DHEA inhibited catecholamine secretion and cytosolic Ca²⁺ ([Ca²⁺]_i) rise coupled with nicotinic acetylcholine receptor (nAChR) without exerting an effect on³H-nicotine binding. In the case of high K⁺ stimulation, DHEA effectively suppressed secretion without affecting [Ca²⁺]₁ rise. Trifluoperazine (TFP), a calmodulin inhibitor, was capable of counteracting the inhibition of DHEA on high K⁺-induced secretions. In permeabilized cells, DHEA suppressed the Ca²⁺-induced secretion. These results suggest that DHEA (a) acts as a channel blocker that suppresses Ca²⁺ influx and subsequent secretions associated with nAChR, or (b) affects the intracellular secretion machinery to suppress high K⁺-induced secretions without affecting the high K⁺-induced [Ca²⁺]_i rise.     

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells

1. Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. 2. In this study trypsin (30-50 micrograms/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. 3. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsin-sensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. 4. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3-10 micrograms/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.     

Relative contribution of the Na(+)/Ca(2+) exchanger, mitochondria and endoplasmic reticulum in the regulation of cytosolic Ca(2+) and catecholamine secretion of bovine adrenal chromaffin cells

The relative importance of mitochondria, the Na(+)/Ca(2+) exchanger (NCX) and the endoplasmic reticulum (ER) in the regulation of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) were examined in bovine chromaffin cells using fura-2 for average [Ca(2+)](i) and amperometry for secretory activity, which reflects the local Ca(2+) concentration near the exocytotic sites. Chromaffin cells were stimulated by a high concentration of K(+) when the three Ca(2+) removal mechanisms were individually or simultaneously inhibited. When the mitochondrial Ca(2+) uptake was inhibited, the [Ca(2+)](i) decayed at a significantly slower rate and the secretory activity was higher than the control cells. The NCX appears to function only in the initial phase of [Ca(2+)](i) decay and when the ER Ca(2+) pump is blocked. Similarly, the ER had a significant effect on the [Ca(2+)](i) decay and on the secretion only when the NCX was blocked. Inhibition of all three mechanisms leads to a substantial delay in [Ca(2+)](i) recovery and an increase in the secretion. The results suggest that the three mechanisms work together in the regulation of the Ca(2+) near the Ca(2+) channels and exocytotic sites and therefore modulate the secretory activity. When Ca(2+) diffuses away from the exocytotic sites, the mitochondrial Ca(2+) uptake becomes the dominant mechanism.     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.     

Lysophosphatidic acid-LPA1 receptor-Rho-Rho kinase-induced up-regulation of Nav1.7 sodium channel mRNA and protein in adrenal chromaffin cells: enhancement of 22Na+ influx, 45Ca2+ influx and catecholamine secretion

In cultured bovine adrenal chromaffin cells, chronic (> or = 24 h) treatment with lysophosphatidic acid (LPA) augmented veratridine-induced 22Na+ influx via Na(v)1.7 by approximately 22% (EC(50) = 1 nmol/L), without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated channel. LPA enhanced veratridine (but not nicotine)-induced 45Ca2+ influx via voltage-dependent calcium channel and catecholamine secretion. LPA shifted concentration-response curve of veratridine for 22Na+ influx upward, without altering the EC(50) of veratridine. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced 22Na+ influx by twofold in non-treated and LPA-treated cells. Whole-cell patch-clamp analysis showed that peak Na+ current amplitude was greater by 39% in LPA (100 nmol/L for 36 h)-treated cells; however, I-V curve and steady-state inactivation/activation curves were comparable between non-treated and LPA-treated cells. LPA treatment (> or = 24 h) increased cell surface [3H]saxitoxin binding by approximately 28%, without altering the K(d) value; the increase was prevented by cycloheximide, actinomycin D, or Ki16425, dioctylglycerol pyrophosphate 8:0 (two inhibitors of LPA(1) and LPA3 receptors), or botulinum toxin C3 (Rho inhibitor), Y27632 (Rho kinase inhibitor), consistent with LPA(1) receptor expression in adrenal chromaffin cells. LPA raised Nav1.7 mRNA level by approximately 37%. Thus, LPA-LPA(1) receptor-Rho/Rho kinase pathway up-regulated cell surface Nav1.7 and Nav1.7 mRNA levels, enhancing veratridine-induced Ca2+ influx and catecholamine secretion.     

Catecholamines-evoked cytosolic ca2+rise in endothelial cells from bovine adrenal medulla

The effects of catecholamines on intracellular Ca²⁺concentrations ([Ca²⁺]_i) in single acutely dissociated bovine adrenal medulla endothelial cells (BAMECs) were measured using the intracellular fluorescent probe Fluo-3 AM. 100 m epinephrine or norepinephrine induced a biphasic [Ca²⁺]_i rise with an initial peak followed by a delayed phase. 10 m phenylephrine (₁-adrenergic agonist) caused a [Ca²⁺]_i rise similar to that evoked by catecholamines. The increase in [Ca²⁺]_i induced by 10 m phenylephrine was reverted by 10 m phenoxybenzamine (-adrenergic antagonist). Neither isoproterenol (-adrenergic agonist) nor clonidine (₂-adrenergic agonist) induced [Ca²⁺]_i rise. The initial peak was insensitive to zero external Ca²⁺ and it was abolished after Ca²⁺ internal storages were emptied by 10 mM caffeine. The delayed phase was reduced to near zero by external Ca²⁺ removal. These results indicate that BAMECs possess ₁-adrenergic receptors associated to both the release of caffeine-sensitive intracellular Ca²⁺ stores and the entry of extracellular Ca²⁺ We suggest that chromaffin cell secretion may activate BAMECs in vivo through an increase in [Ca²⁺]_i which could induce the secretion of vasoactive factors allowing a rapid entry of hormones into the circulation. (Mol Cell Biochem 000: 000-000,1999)     

Mechanism of Na+ deprivation-induced catecholamine secretion from freshly isolated bovine adrenal chromaffin cells

We have studied the mechanism of Na+ deprivation-induced catecholamine secretion from freshly isolated bovine adrenal chromaffin cells. Na+ deprivation-induced catecholamine secretion depended on free extracellular Ca2+ concentrations and was almost parallel to 45Ca2+ influx into the cells under various experimental conditions. Furthermore, Na+ deprivation-induced 45Ca2+ influx and catecholamine secretion were actually induced by a relative Na+ concentration gradient across the plasma membrane, but not by simple omission of Na+ from the medium. These results indicate that the deprivation of Na+ from the medium changes the relative Na+ gradient across the plasma membrane and results in Ca2+ influx via a reverse mode of Na(+)-Ca(2+) exchange rather than by inducing Ca2+ entry through Ca2+ channels by eliminating the competition between extracellular Na+ and Ca2+.     

Enhancement by cytochalasin B of ouabain-stimulated catecholamine secretion from cultured bovine adrenal chromaffin cells: possible relation to alteration in Na+/K+-pump activity

1. Catecholamine secretion evoked by ouabain from cultured bovine adrenal chromaffin cells has previously been shown to be markedly enhanced by pretreatment of the cells with cytochalasin B (Morita et al., 1988). To elucidate a possible mechanism of this enhancement, the stimulatory action of ouabain on Ca2+ influx as well as catecholamine secretion was then examined in the cells pretreated with or without cytochalasin B. The effect of cytochalasin B pretreatment on the inhibitory action of ouabain on the Na+/K+ pump was also examined by measuring 86Rb+ uptake into the cells. 2. Pretreatment of the cells with cytochalasin B caused enhancement of ouabain-induced catecholamine secretion, and this enhancement was accompanied by the elevation of ouabain-stimulated 45Ca2+ uptake into the cells. The inhibitory action of ouabain on 86Rb+ uptake was significantly enhanced by pretreatment of the cells with cytochalasin B under the same conditions. 3. These results indicate that the enhancement of ouabain-induced catecholamine secretion caused by cytochalasin B pretreatment may be due to the increase in ouabain-stimulated Ca2+ influx into the cells and, furthermore, suggest the possibility that this increase in Ca2+ influx may be attributed to the potentiation of the inhibitory action of ouabain on the Na+/K+ pump in the adrenal chromaffin cell. Thus, the present study provides an evidence for a possible role of microfilaments as one of the intrinsic factors modulating the plasma membrane functions.     

A comparison of bradykinin,angiotensin II and muscarinic stimulation of cultured bovine adrenal chromaffin cells

Bradykinin, angiotensin II and a mascarnic agonist, acetyl-B-methacholine (methacholine) were all found to elict catecholamine release from cultured bovine adrenal chromaffin cells. Bradykinin was the most potent of these secretagogues and methacholine the weakest, with angiotenin II intermediate in efficacy. All three secretagogues were much less effective than nicotinic stimulation. The three secretagogues all produced a rise in cytoplasmic free calcium concentration ([Ca²⁺]_i), measured with the fluorescent indicator fura2, which was partially independent of external calcium. In the case of bradykinin the full rise in ([Ca²⁺]_i) may involve a component of calcium entry in addition to release of calcium from an internal store. Secretion was also found to be partially independent of external calcium. The different efficacies of the three secretagogues in elicting secretion were correlated with the rise in ([Ca²⁺]_i) produced. The differeing efficacies of the three secretagogues may be due to the extent of release of calcium from an intracellular store which itself is less effective in eliciting secretion than a rise in [Ca²⁺]_i following calcium entry due to nicotine. Bradykinin also stimulates calcium entry, and this may increase the efficacy of the initial rise in [Ca²⁺]_i. Treatment with pertussis toxin resulted in an enhancement of secretion in response to all of the secretagogues.Abbreviations ([Ca²⁺]_i) cytoplasmic free calcium concentration - EGTA ethylene glycol bis (-amino ethyl ether)-N,N,N,N,-tetraacetic acid - Hepes 4-(2-hydroxy ethyl)-1-piperazinethanesulphonic acid - TPA 12-O-tetradecanoylphorbol-13-acetate - DAG diacyl glycerol - IP₃ inositol-1,4,5-trisphosphate - PIP₂ phosphatidylinositol-4,5-bisphosphate     

Effect of calmidazolium and phorbol ester on catecholamine secretion from adrenal chromaffin cells

Carbamylcholine-stimulated catecholamine release from adrenal chromaffin cells was completely inhibited by pretreatment of the cells for 10 min with 1 μM calmidazolium. Catecholamine release due to 55 mM K⁺ and ionophore A23187 was also inhibited by calmidazolium but less effectively than release due to carbamylcholine. Inhibition of release appeared to be due to an effect of calmidazolium on a step distal to Ca²⁺ entry, since the carbamylcholine-stimulated rise in the concentration of intracellular free calcium, monitored using quin-2, was unaffected by calmidazolium. The possibility was considered that calmidazolium inhibited secretion through an effect on protein kinase C rather than calmodulin. However, the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), had no demonstrable effect on catecholamine release, arguing against a significant role for protein kinase C in secretion from adrenal chromaffin cells. These results give further support to the notion that calmodulin plays a role in the secretory process in chromaffin cells.     

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.     

Primary structure of an agonist binding subunit of the nicotinic acetylcholine receptor from bovine adrenal chromaffin cells

Activation by acetylcholine of a nicotinic acetylcholine receptor on the membrane of bovine chromaffin cells leads to membrane depolarization and to the subsequent triggering of catecholamine secretion. It is evident that acetylcholine receptors play a central role in the initial phase of the secretion process and, therefore, an extensive characterization of their molecular components and properties is of fundamental interest. With this intention, we have screened bovine adrenal medullary cDNA libraries with a probe coding for a fragment of the rat muscle acetylcholine receptor subunit. Several cDNA clones were isolated. The longest cDNA had an open reading frame encoding a 495-amino acid protein with a molecular weight of 56,911. The deduced primary structure contains features that indicate that the encoded protein is an or acetylcholine binding subunit, and, in fact, it manifests significant sequence similarity to previously cloned subunits. Sequence identity is particularly high with the 3 subunit, which is expressed in the rat pheochromocytoma PC12 cell line and in several brain areas, and consequently, it is considered a component of a neuronal acetylcholine receptor. Accordingly, the present results suggest that the agonist binding subunit of the nicotinic acetylcholine receptor from bovine chromaffin cells is an 3-type subunit, corroborating previous immunological and pharmacological evidence for the presence of a neuronal nicotinic receptor in chromaffin cells.Abbreviations used nAChR nicotinic acetylcholine receptor - SDS sodium dodecyl sulfate - SSC 0.15 M NaCl and 0.015 M sodium citrate - kb kilobases - bp base pairs     

Enhancement of Ca2+-induced catecholamine release by the phorbol ester TPA in digitonin-permeabilized cultured bovine adrenal chromaffin cells

The phorbol ester, 4 beta-phorbol 12-myristate acetate (TPA), increased the extent of catecholamine release induced by Ca2+, without affecting the basal release response in digitonin-permeabilized chromaffin cells. This finding is consistent with the hypothesis that protein kinase C has a role to play in stimulus-secretion coupling in the bovine adrenal medullary chromaffin cell.     

Expression of dimeric and tetrameric acetylcholinesterase isoforms on the surface of cultured bovine adrenal chromaffin cells

Acetylcholinesterase is a highly polymorphic enzyme, which can be anchored to the cell surface through several different mechanisms. Dimeric (G2) acetylcholinesterase isoforms are attached by a glycosylphosphatidyl-inositol (GPI) linkage, whereas tetrameric (G4) forms are linked through a 20 kilodalton hydrophobic subunit. Although cells of haemopoietic origin contain large amounts of G2 GPI-linked acetylcholinesterase, most tissues express only trace amounts of this isoform. We examined the expression of acetylcholinesterase isoforms in cultured bovine adrenal medullary chromaffin cells. Two major isoforms (G2 and G4) were identified on the cell surface. The G2 isoform, which accounted for approximately half the cell-surface enzyme activity, was linked to the membrane through a GPI anchor. After treatment with diisopropylfluorophosphate to completely inhibit cellular acetylcholinesterase, the G4 isoform was found to be resynthesised and transported to the cell surface more rapidly than the G2 isoform. As the addition of GPI anchors is known to be a very rapid step, this finding suggested that the G2 and G4 isoforms might be transported to the cell surface by two different mechanisms. This conclusion was supported by results from subcellular fractionation experiments. The ratio of G4/G2 membrane-bound acetylcholinesterase varied between different subcellular fractions. The membrane-bound G2 isoform was greatly enriched in a high-speed “microsomal” fraction. G4 acetylcholinesterase is known to be actively secreted by chromaffin cells in culture. Although the G4 isoform was present on the cell surface, most of the secreted enzyme was derived from an intracellular pool. Thus, it is unlikely that the cell-surface G4 isoform contributes significantly to the pool of secreted enzyme. Instead, the expression of two different membrane-bound isoforms may provide a means by which chromaffin cells can target the enzyme to different locations on the cell surface. © 1994 Wiley-Liss, Inc.     

Binding of prostaglandin E2 to cultured bovine adrenal chromaffin cells and its effect on catecholamine secretion

We have previously shown that plasma membranes from adrenal medulla possess specific high-affinity binding sites for prostaglandins (PGs) E1 and E2. We have now investigated the binding of PGE2 to intact bovine adrenal chromaffin cells and the effects of prostaglandins on the release of catecholamines from these cells. Adrenal chromaffin cells specifically bound PGE2 with a dissociation constant of 2 nM and a concentration of about 40,000 binding sites per cell. Low concentrations of PGE2 inhibited the nicotine-stimulated release of catecholamines from these cells. The effect of PGE2 was biphasic, the maximal inhibitory effect being observed at a concentration of between 1 and 10 nM. Higher concentrations (1 microM) of PGE2 had minimal inhibitory effects on nicotine-evoked noradrenaline release, but instead had a direct stimulatory effect in the absence of cholinergic agonists. Although the stimulatory effects of high concentrations of PGE2 were reproducibly observed in all cell preparations, only about one-half of the cultures tested responded to the inhibitory effects of this prostaglandin. It is possible that PGE2 plays a modulatory role in the regulation of catecholamine secretion from the adrenal medulla.     

Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells

This study was made to explain the mechanisms for the effects of exposure to a time varying 1.51 T magnetic field on the intracellular Ca(2+) signaling pathway. The exposure inhibited an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine chromaffin cells induced by addition of bradykinin (BK) to a Ca(2+) free medium. The exposure did not change BK induced production of inositol 1,4,5-trisphosphate (IP(3)). [Ca(2+)](i) was markedly increased in IP(3) loaded cells, and this increase was inhibited by the magnetic field exposure. A similar increase in [Ca(2+)](i) by other drugs, which stimulated Ca(2+) release from intracellular Ca(2+) stores, was again inhibited by the same exposure. However, transmembrane Ca(2+) fluxes caused in the presence of thapsigargin were not inhibited by the magnetic field exposure in a Ca(2+) containing medium. Inhibition of the BK induced increase in [Ca(2+)](i) by the exposure for 30 min was mostly recovered 1 h after exposure ended. Our results reveal that the magnetic field exposure inhibits Ca(2+) release from intracellular Ca(2+) stores, but that BK bindings to BK receptors of the cell membrane and intracellular inositol IP(3) production are not influenced.     

Role of protein phosphatase 2C from bovine adrenal chromaffin cells in the dephosphorylation of phospho-serine 40 tyrosine hydroxylase

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. It is dephosphorylated by protein phosphatase (PP) 2A and PP2C. In this study we used a fixed amount of bacterially expressed rat TH (5 microM), phosphorylated only at serine 40 (pSer40TH), to determine the PP activities against this site that are present in extracts from the bovine adrenal cortex, adrenal medulla, adrenal chromaffin cells and rat striatum. We found that PP2C was the main TH phosphatase activity in extracts from the adrenal medulla and adrenal chromaffin cells. In adrenal cortex extracts PP2C and PP2A activities toward pSer40TH did not differ significantly. PP2A was the main TH phosphatase activity in extracts from rat striatum. Kinetic studies with extracts from adrenal chromaffin cells showed that when higher concentrations of pSer40TH (> 5 microM) were used the activity of PP2C increased more than the activity of PP2A. PP2C was maximally activated by 1.25 mM Mn2+ and by 5 mM Mg2+ but was inhibited by calcium. Our data suggest a more important role for PP2C than was previously suggested in the dephosphorylation of serine 40 on TH.     

Synergistic actions of Ca2+ and the phorbol ester TPA on K+-induced catecholamine release from bovine adrenal chromaffin cells

Enhancement of Ca2+-dependent high K+-evoked catecholamine secretion was observed after pretreatment of cultured bovine adrenal chromaffin cells with the phorbol ester 4B-phorbol 12-myristate 13-acetate (TPA) in the absence of added extracellular Ca2+. This effect of TPA was not reproduced when the secretagogues acetylcholine, nicotine, or veratrine were substituted for high K+. The implications of these results are discussed in relation to the role of protein kinase C in stimulus-secretion coupling in the chromaffin cell.     

乙酰胆碱和A23187对离体大鼠肾上腺髓质细胞肾上腺素分泌的作用

通过胆碱能激动剂乙酰胆碱及离子诱导剂A23187（以下简称激动剂）作用于分离的肾上腺髓质细胞,以引起离体细胞的刺激－分泌耦联过程,运用细胞立体形态计量法计算分泌过程中的嗜铬颗粒数目的变化,运用电镜X射线显微分析法测量分泌过程中嗜铬颗粒内钙含量的变化,并运用高 液相色谱分析法测定离体细胞在激动剂作用后的肾上腺素分泌情况,结果发现,分离的肾上腺髓质细胞嗜铬颗粒内钙含量在激动剂作用10min时有明显下降,颗粒数目在激动剂作用过程中呈缓慢下降趋势,而细胞悬液中的肾上腺素含量在激动剂作用20min以后有明显的升高,激动剂作用引起的离体肾上腺髓质的细胞分泌时颗粒内钙含量的下降早于颗粒数目减少或肾上腺素升高,提示颗粒释放的Ca^2 可能是引起细胞分泌的原因之一。