Effect of quinine on the release of catecholamines from bovine cultured chromaffin cells.

1. The effects of quinine on catecholamine release from cultured bovine chromaffin cells were studied. 2. Quinine (25-400 microM) produced a dose-related inhibition of catecholamine release in response to depolarizing concentrations (12.5-50 mM) of K+. 3. The inhibition of the secretory response to high K+ produced by quinine decreased with the increase in the extracellular concentration of Ca2+. 4. Stimulation of cultured chromaffin cells with 50 mM K+ produced a significant increase in Ca2+ influx. In the presence of 100 microM quinine a 54% inhibition of the K(+)-induced Ca2+ influx was observed. 5. Quinine treatment of chromaffin cell cultures produced a small but significant decrease in membrane resting potential and a less pronounced depolarization in response to 50 mM K+. 6. The results suggest that the inhibition of the K(+)-evoked release of catecholamines produced by quinine is at least partly due to a decrease in Ca2+ influx. Ca2+ influx is lower because quinine reduces the sensitivity of the membrane potential to changes in extracellular K+ but direct effects of quinine on Ca2+ channels cannot be excluded.     

Effects of inhibitors of arachidonic acid metabolism on calcium uptake and catecholamine release in cultured adrenal chromaffin cells

The possibility that arachidonic acid metabolism is involved in the secretory process in cultured adrenal chromaffin cells was investigated by studying the effects of lipoxygenase inhibitors and cyclooxygenase inhibitors on 45Ca2+ uptake and catecholamine release. Lipoxygenase inhibitors, which have different chemical structures, such as nordihydroguaiaretic acid (NDGA), 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline (BW755C) and 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861) all prevented the catecholamine release evoked by carbamylcholine and high K+. In contrast, cyclooxygenase inhibitors, such as aspirin and indomethacin failed to inhibit the carbamylcholine-evoked catecholamine release. Lipoxygenase inhibitors also inhibited 45Ca2+ uptake into the cells stimulated by carbamylcholine and high K+. Lipoxygenase inhibitors inhibited 45Ca2+ uptake and catecholamine release with similar potency. Slightly higher concentrations of lipoxygenase inhibitors were required to inhibit high K+-evoked effects compared to those evoked by carbamylcholine. The inhibitory effects of these inhibitors on carbamylcholine-evoked catecholamine release was different in its nature from the inhibitory effect of verapamil, a blocker of the Ca2+ channel, and was not due to a competitive antagonism at cholinergic receptor site. Moreover, these lipoxygenase inhibitors did not inhibit the binding of [3H]nitrendipine to chromaffin cell homogenate. The data suggest that lipoxygenase inhibitors prevent the catecholamine release from cultured adrenal chromaffin cells by blocking Ca2+ uptake. It might be possible that lipoxygenase product(s) is involved in the Ca2+ translocation system in these cells.     

Studies on the uptake and release of propranolol and the effects of propranolol on catecholamines in cultures of bovine adrenal chromaffin cells

Uptake and release of [3H]l-propranolol and the effects of propranolol on the uptake and release of [3H]norepinephrine were studied in cultures of isolated bovine adrenal chromaffin cells. [3H]l-Propranolol uptake increased with increasing [3H]l-propranolol concentration from 10(-7) M to 10(-3) M and was not saturable in this concentration range. [3H]l-Propranolol uptake was equally inhibited by l- and d-propranolol, indicating that the uptake is not stereoselective. [3H]l-Propranolol uptake differed from [3H]norepinephrine uptake in two respects: [3H]l-propranolol uptake was 44-50 times greater than [3H]norepinephrine uptake at early non-equilibrium time periods, and [3H]l-propranolol uptake was not Na+ dependent and was not inhibited by desipramine, indicating that [3H]l-propranolol is not taken up by the biogenic amine transport system. In cells preloaded with [3H]l-propranolol, two agents, veratridine and tyramine, stimulated an increased release of [3H]l-propranolol into the medium. However, veratridine-induced [3H]l-propranolol release was inhibited only slightly by the Na+ channel blocker tetrodotoxin, and tyramine-induced [3H]l-propranolol release was not inhibited by desipramine. In addition, K+, carbachol and the physiological mediator of adrenal catecholamine release, acetylcholine, failed to evoke [3H]l-propranolol release. Therefore, it is unlikely that propranolol is released in response to physiological stimulation of adrenal chromaffin cells in animals administered propranolol in vivo. l-Propranolol inhibited [3H]norepinephrine uptake by chromaffin cells with an IC50 for l-propranolol of 5 X 10(-6) M; d-propranolol was equally potent for this effect at lower propranolol concentrations. By themselves, neither l- nor d-propranolol had any effect on [3H]norepinephrine release from the cells. However, l-propranolol inhibited carbachol-induced [3H]norepinephrine release with an IC50 for l-propranolol of 5 X 10(-7) M to 10(-6) M. At these lower concentrations, d-propranolol had no effect on carbachol-induced [3H]norepinephrine release, indicating that the inhibition by l-propranolol may be mediated via beta-adrenoceptors on chromaffin cells.     

Monensin-induced influx of 22Na and the release of catecholamines in cultured bovine adrenal medulla cells and isolated chromaffin granules

In cultured bovine adrenal medulla cells, monensin caused the release of catecholamines simultaneous with the influx of 22Na to the cells. The release of catecholamines by monensin was dependent on Na but not on Ca in the medium. Release of catecholamines and the influx of 22Na caused by monensin were not inhibited by tetrodotoxin. Monensin did not cause the release of dopamine beta-hydroxylase from the cells showing that monensin caused the release of catecholamines by a nonexocytotic mechanism. Similarly, in isolated chromaffin granules, monensin caused Na-dependent release of catecholamines, simultaneously with the influx of 22Na to the granules. Basing on these findings, monensin seems to cause a nonexocytotic release of catecholamines by acting as an Na ionophore both at cell membranes and chromaffin granule.     

Effect of noncompetitive nicotinic receptor blockers on catecholamine release from cultured adrenal chromaffin cells.

Nicotinic acetylcholine receptors are found on both muscle tissue and neuronal tissue, including adrenal chromaffin cells. Certain drugs, such as the noncompetitive ion-channel blockers and the vinca alkaloids, have been demonstrated to interact with muscle-type nicotinic receptor-associated ion channels. The objectives of these studies were: (1) to determine the effects of the noncompetitive blockers of the nicotinic receptor-associated ion-channel drugs on adrenal chromaffin cells, and (2) to establish whether the vinca alkaloids and the ion-channel drugs have similar actions on adrenal nicotinic receptors. The ion-channel drugs, adiphenine, tetracaine, quinacrine, amantadine, lidocaine and procaine, inhibited receptor-stimulated catecholamine release from cultured adrenal chromaffin cells in a concentration-dependent manner (IC50s: 2, 4, 6, 41, 55 and 87 microM, respectively). The inhibitory effects of these drugs appeared to be noncompetitive. These drugs had little or no effects on catecholamine release stimulated by depolarizing concentrations of K+ or on basal release. Our results demonstrate that the ion-channel blockers interact with adrenal nicotinic receptors in a manner similar to their interaction with muscle-type nicotinic receptors and they interfere with adrenal receptor function in a manner similar to the vinca alkaloids.     

Increase in catecholamine release and 45Ca2+ uptake induced by GABA in cultured bovine adrenal chromaffin cells

The role of Ca2+ in GABA-evoked catecholamine (CA) release from adrenal medulla was investigated in primary cultures of bovine adrenal chromaffin cells. GABA facilitated the 45Ca2+ uptake associated with the increase of Ca release in cultured bovine adrenal chromaffin cells. The effects of GABA on both 45Ca2+ uptake and CA release were blocked by bicuculline and picrotoxin. Nifedipine reduced the 45Ca2+ uptake and CA release induced by GABA. These data support our previous suggestion that the activation of GABA receptors on adrenal chromaffin cells facilitates the Ca2+ influx through voltage-sensitive Ca2+ channels, leading to the release of CA.     

Involvement of phosphoinositide metabolism in GABA-induced catecholamine release from cultured bovine adrenal chromaffin cells

The effects of GABA on catecholamine release and phosphoinositide metabolism were studied in cultured bovine adrenal chromaffin cells. GABA and muscimol, a specific agonist for the GABAA receptor, each evoked a gradual secretion of catecholamines from the cells in the presence of ouabain, an inhibitor of Na+, K(+)-ATPase. This release was inhibited by bicuculline, a specific antagonist for the GABAA receptor, or by picrotoxin, a blocker of GABA-gated Cl- channels, and was potentiated by diazepam or pentobarbital. GABA or muscimol induced a concentration-dependent formation of inositol phosphates. This accumulation of inositol phosphates was also inhibited by bicuculline, picrotoxin or removal of extracellular Ca2+, and also potentiated by diazepam and pentobarbital. Nicardipine suppressed GABA-induced catecholamine release in the presence of ouabain and accumulation of inositol phosphates, while verapamil, diltiazem, and omega-conotoxin failed to inhibit these responses to GABA. The phosphoinositide-specific phospholipase C inhibitor neomycin also inhibited both GABA-induced accumulation of inositol phosphates and stimulation of catecholamine release in the presence of ouabain. These results taken together indicate that GABA evoked catecholamine release from the chromaffin cells in the presence of ouabain by stimulation of phosphoinositide metabolism in a Ca2(+)-sensitive manner via activation of GABAA receptor-coupled Cl- channels.     

Enhancement by GABA of the stimulation-evoked catecholamine release from cultured bovine adrenal chromaffin cells

Summary The possible involvement of GABAergic mechanisms in the catecholamine (CA) release from adrenal medulla was investigated in a primary culture of bovine adrenal chromaffin cells. GABA elicited CA release and enhanced acetylcholine (ACh)-, excess K⁺-and veratridine-evoked CA release. Muscimol, a selective GABA_A receptor agonist, mimicked the action of GABA on CA release. On the other hand, baclofen, a GABA_B receptor agonist, failed to affect basal or evoked CA release. Furthermore, bicuculline and picrotoxin blocked the enhancement by GABA of veratridine-evoked CA release without affecting basal CA release and CA release evoked by veratridine. In Ca²⁺-free medium, GABA failed to affect basal and caffeine-evoked CA release. ACh-evoked CA release was slightly reduced by bicuculline, whereas excess K⁺-evoked CA release was not, suggesting the involvement of endogenous GABA in CA release evoked by ACh. These results suggest a facilitatory modulation by GABA of basal and evoked release of CA from bovine adrenal medulla through GABA_A receptor-mediated mechanisms. Send offprint requests to A. Tsujimoto at the above address     

Subacute nicotine exposure in cultured cerebellar cells increased the release and uptake of glutamate

Cerebellar granule and glial cells prepared from 7 day-old rat pups were used to investigate the effects of sub-acute nicotine exposure on the glutamatergic nervous system. These cells were exposed to nicotine in various concentrations for 2 to 10 days in situ. Nicotine-exposure did not result in any changes in cerebellar granule and glial cell viability at concentrations of up to 500 microM. In cerebellar granule cells, the basal extracellular levels of glutamate, aspartate and glycine were enhanced in the nicotine-exposed granule cells. In addition, the responses of N-methyl-D-aspartate (NMDA)-induced glutamate release were enhanced at low NMDA concentrations in the nicotine-exposed granule cells. However, this decreased at higher NMDA concentrations. The glutaminase activity was increased after nicotine exposure. In cerebellar glial cells, glutamate uptake in the nicotine-exposed glial cells were either increased at low nicotine exposure levels or decreased at higher levels. The inhibition of glutamate uptake by L-trans-pyrollidine-2,4-dicarboxylic acid (PDC) was lower in glial cells exposed to 50 microM nicotine. Glutamine synthetase activity was lower in glial cells exposed to 100 or 500 microM of nicotine. These results indicate that the properties of cerebellar granule and glial cells may alter after subacute nicotine exposure. Furthermore, they suggest that nicotine exposure during development may modulate glutamatergic nervous activity.     

Evidence for a dihydropyridine-sensitive and conotoxin-insensitive release of noradrenaline and uptake of calcium in adrenal chromaffin cells.

1. It has been suggested that neuronal voltage-sensitive calcium channels (VSCC) may be divided into dihydropyridine (DHP)-sensitive (L) and DHP-insensitive (N and T), and that both the L and the N type channels are attenuated by the peptide blocker omega-conotoxin. Here the effects of omega-conotoxin on release of noradrenaline and uptake of calcium in bovine adrenal chromaffin cells were investigated. 2. Release of noradrenaline in response to 25 mM K+, 65 mM K+, 10 nM bradykinin or 10 microM prostaglandin E1 was not affected by omega-conotoxin in the range 10 nM-1 microM. 3. 45Ca2+ uptake stimulated by high K+ and prostaglandin was attenuated by 1 microM nitrendipine and enhanced by 1 microM Bay K 8644; these calcium fluxes were not modified by 20 nM omega-conotoxin. 4. With superfused rat brain striatal slices in the same medium as the above cell studies, release of dopamine in response to 25 mM K+ was attenuated by 20 nM omega-conotoxin. 5. These results show that in these neurone-like cells, release may be effected by calcium influx through DHP-sensitive but omega-conotoxin-insensitive VSCC, a result inconsistent with the suggestion that omega-conotoxin blocks both L-type and N-type neuronal calcium channels.     

Tetrodotoxin-insensitive Na+ channel activator palytoxin inhibits tyrosine uptake into cultured bovine adrenal chromaffin cells.

The effects of the tetrodotoxin-insensitive Na+ channel activator palytoxin on both the secretion of endogenous catecholamines and the formation of 14C-catecholamines from [14C]tyrosine were examined using cultured bovine adrenal chromaffin cells. Palytoxin was shown to cause the stimulation of catecholamine secretion in a concentration-dependent manner. However, this toxin caused the reduction rather than the stimulation of 14C-catecholamine formation at the same concentrations. Palytoxin failed to cause any alteration in the activity of tyrosine hydroxylase prepared from bovine adrenal medulla. Furthermore, the uptake of [14C]tyrosine into the cells was shown to be inhibited by this toxin under the conditions in which the suppression of 14C-catecholamine formation was observed, and this inhibitory action on tyrosine uptake was closely correlated with that on catecholamine formation. The inhibitory action of palytoxin on tyrosine uptake into the cells was observed to be noncompetitive, and this effect was not altered by the removal of Na+ from the incubation mixture. These results suggest that palytoxin may be able to inhibit the uptake of [14C]tyrosine into the cells, resulting in the suppression of 14C-catecholamine formation, probably through its direct action on the plasma membranes of bovine adrenal chromaffin cells.     

Modulation of calcium uptake and D-aspartate release by GABAB receptors in cultured cerebellar granule cells

(-)Baclofen, a GABAB receptor agonist, and GABA attenuated by 60% the high K+-evoked 45Ca2+ uptake into cultured cerebellar granule cells with an EC50 of 110 +/- 18 nM and 2.4 +/- 0.2 microM, respectively. The attenuation by baclofen of 45Ca2+ uptake was stereospecific and the effect of GABA was unaffected by bicuculline. Moreover, muscimol, a GABAA receptor agonist did not affect the K+-evoked 45Ca2+ uptake. (-)Baclofen and GABA also decreased the K+-evoked and calcium-dependent release of preloaded [3H]D-aspartate from granule cells; however, their potency and efficacy appeared to be less than those for inhibiting the 45Ca2+ uptake. (+)Baclofen and muscimol failed to change this K+-evoked release. The release of [3H]D-aspartate induced by the calcium ionophore A23187 was unaffected by (-)-baclofen. The K+-evoked release of [3H]D-aspartate was effectively inhibited by nimodipine, a voltage sensitive calcium channel blocker. The results suggest that GABAB receptor in cultured cerebellar granule cells plays a crucial role in modulating the uptake of calcium and release of the excitatory transmitter. Moreover, these two effects mediated by GABAB receptor activation may be casually related.     

丹皮酚对体外培养乳鼠心肌细胞~(45)Ca摄取的影响

通过测定体外培养乳鼠心肌细胞~(45)Ca摄取及其搏动频率的变化,观察丹皮酚对心肌细胞Ca~(2+)内流的影响。结果50～400μg/ml的丹皮酚,能显著降低心肌细胞搏动频率,对心肌细胞快相(5min)和慢相(120min)~(45)Ca摄取均有显著抑制作用,且400μg/ml丹皮酚与10μmol/L的维拉帕米的作用相似。提示丹皮酚能抑制心肌细胞的Ca~(2+)内流,可能与阻滞慢钙通道有关。     

Prostaglandin E2-induced arachidonic acid release and catecholamine secretion from cultured bovine adrenal chromaffin cells.

We recently reported that prostaglandin E2 (PGE2) and arachidonic acid (AA) each induced a gradual secretion of catecholamines from cultured bovine adrenal chromaffin cells in the presence of ouabain by stimulation of phosphoinositide metabolism. In the present study, we examined the relationship between phospholipase A2 and C activation and catecholamine secretion by PGE2 in chromaffin cells. The phospholipase A2 inhibitors p-bromophenacyl bromide and mepacrine did not affect the basal and ouabain-induced release, but dose-dependently blocked PGE2-evoked phosphoinositide metabolism and the consequent catecholamine release at an IC50 value of 3 microM. PGE2 induced rapid hydrolysis of [3H]AA from prelabeled phospholipid pools: the release of [3H]AA could be detected at as early as 15 sec and reached a plateau after 1 min. While the phospholipase C inhibitor neomycin did not inhibit PGE2-induced AA release, phospholipase A2 inhibitors dose-dependently inhibited it at IC50 values comparable to those for catecholamine release. Pretreatment of intact cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, but not with pertussis toxin, prevented AA release by PGE2. These results demonstrate that PGE2 activates phospholipase A2 as well as phospholipase C in a pertussis toxin-insensitive manner and suggest that the released arachidonic acid may be involved in PGE2-induced catecholamine release from chromaffin cells.     

Effects of collagenase on the release of [3H]-noradrenaline from bovine cultured adrenal chromaffin cells.

Bovine isolated adrenal chromaffin cells maintained in culture at 37 degrees C for 1-7 days become polygonal and bipolar, with typical varicosity-like extensions. Catecholamine levels and dopamine beta-hydroxylase activity decreased after 24-48 h of culture, but recovered to normal levels 3-7 days later. Incubation of 1-7 day-old cells in the presence of increasing concentrations of [3H]-noradrenaline (3.91 to 125 nM) resulted in the retention by the cells of amounts of radioactivity directly proportional to the amine present in the media. One day-old cells took up and retained only one third of the radioactivity found in 2-7 day-old cells. The addition of collagenase to cultured cells caused a decrease in the uptake of tritium. However, the enzyme treatment did not affect the amine taken up by the cell before collagenase treatment. Release of tritium from cultured cells evoked by nicotine, acetylcholine (ACh) or 59 mM K+ was very poor in 24 h-old cells; the secretory response to nicotine, ACh or K+ was dramatically increased after 2-7 days of culture. Bethanecol did not cause any secretory response. When treated with collagenase, cultured cells which had recovered fully their secretory response, lost again the ability to release tritium evoked by ACh or nicotine. However, the responses to high K+, veratridine or ionophore X537A were not affected. The nicotinic response was recovered two days after collagenase treatment. The data suggest that the use of collagenase to disperse the adrenomedullary tissue during the isolation procedure might be responsible for the lost secretory response of young cultured chromaffin cells. Since collagenase specifically impairs the nicotinic cholinoceptor-mediated catecholamine release, it seems likely that the enzyme is exerting its action on the ACh receptor complex. It is unlikely that either voltage-sensitive Na+ or Ca2+ channels are affected by collagenase as the responses induced by high K+ or veratridine were unaffected by this enzyme.