Differential effects of FK506 and cyclosporin A on catecholamine release from bovine adrenal chromaffin cells

1 The effects of the immunosuppressants, tacrolimus (FK506) and cyclosporin A (CsA), on catecholamine (CA) release were examined in cultured bovine adrenal chromaffin cells. 2 In intact cells, FK506 (1-30 microM) inhibited CA release stimulated by acetylcholine (ACh; 100 microM), 1,1-dimethyl-4-phenyl-piperazinium (DMPP, 10 microM) or high K+ (40 mM). CsA (1-30 microM) had a little inhibitory effect on the ACh- or DMPP-stimulated CA release, whereas it enhanced the high K(+)-stimulated CA release. 3 In beta-escin-permeabilized cells, FK506 inhibited CA release stimulated by Ca2+ (1 and 10 microM) in the presence and absence of MgATP (2 mM). CsA induced CA release under Ca(2+)-free condition and enhanced the Ca(2+)-stimulated CA release in the presence and absence of MgATP. 4 It is known that the Ca(2+)-dependent exocytosis involves at least two distinct steps, ATP-requiring priming stage and ATP-independent fusion step in adrenal chromaffin cells. Therefore, it is suggested that FK506 inhibits the Ca(2+)-dependent exocytosis probably at the fusion step whereas CsA induces CA release from bovine adrenal chromaffin cells.     

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.     

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.     

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.     

The effects of neosurugatoxin on evoked catecholamine secretion from bovine adrenal chromaffin cells.

1. The effect of neosurugatoxin (NSTX), a toxin from the Japanese ivory mollusc (Babylonia japonica), on the nicotinic response of bovine adrenal chromaffin cells was examined. 2. NSTX inhibited acetylcholine- and nicotine-induced catecholamine secretion from the cultured cells with an IC50 against 5 microM nicotine of 30 nM. 3. This inhibitory effect was reversible and independent of the presence of agonist. 4. NSTX had no effect on the catecholamine release from cultured cells evoked by 50 mM K+, or 1 microM histamine. 5. NSTX had no effect on the stimulation of phosphatidylinositol metabolism evoked by 100 microM muscarine. 6. These results suggest NSTX may be useful as a nicotinic receptor probe in tissues such as the adrenal and sympathetic ganglia where alpha-bungarotoxin is ineffective.     

Inhibitory effects of caffeine on secretagogue-induced catecholamine secretion from adrenal chromaffin cells of the guinea-pig.

1. The inhibitory action of caffeine on catecholamine secretion induced by secretagogues was investigated in perfused adrenal glands and dispersed chromaffin cells of the guinea-pig. 2. Caffeine (10 mM) caused a reversible inhibition of catecholamine secretion evoked by acetylcholine (ACh, 50 microM), KCl (56 mM, high K+) and veratridine (100 microM) and that induced by muscarinic receptor activation in the absence of extracellular Ca2+ in perfused adrenal glands. 3. In dispersed chromaffin cells, caffeine caused a dose-dependent inhibition of the secretory responses to 100 microM ACh and veratridine. Forskolin (30 microM), dibutyryl cyclic AMP (1 mM) and 8-bromo cyclic AMP (1 mM) did not mimic the action of caffeine. 4. In the voltage-clamp, whole-cell recording mode (at a holding potential of -60 mV or -70 mV), ACh (100 microM) evoked an inward current, and depolarizing pulses elicited inward Na+, Ca2+ and outward K+ currents. All these responses were partially inhibited by caffeine (20 mM). 5. ACh rapidly increased the intracellular concentration of Ca2+ ([Ca2+]i) in fura-2-loaded cells in either the presence or the absence of external Ca2+, though its magnitude was decreased by about 50% in Ca(2+)-free conditions. Caffeine (20 mM) inhibited these ACh-induced increases in [Ca2+]i. 6. In permeabilized chromaffin cells, caffeine (20 mM) caused an inhibition of catecholamine secretion evoked by Ca2+ (10 microM). 7. These results suggest that caffeine inhibits evoked catecholamine secretion through mechanisms such as the blockade of voltage-dependent Na+ and Ca2+ currents and ACh receptor current, and reduction of the release of intracellularly stored Ca2+ and/or Ca(2+)-sensitivity of the secretory apparatus.     

(+)-isradipine but not (-)-Bay-K-8644 exhibits voltage-dependent effects on cat adrenal catecholamine release.

1. Catecholamine release from cat adrenal glands perfused at a high rate (4 ml min-1) at 37 degrees C with polarizing (1.2 or 5.9 mM K+) or depolarizing (17.7, 35, 59 or 118 mM K+) solutions, was triggered by 5 or 10 s pulses of Ca2+ (0.5 or 2.5 mM) in the presence of various concentrations of K+. 2. In polarized glands, secretion was greater the higher the K+ concentration present during the secretory K+/Ca2+ test pulse. Thus, in 17.7 mM K+, catecholamine released was 162 +/- 27 ng per pulse, while in 118 mM K+ secretion rose to 1839 +/- 98 ng per pulse. In depolarized glands, secretion reached a peak of around 1000 ng per pulse in 35-59 mM K+; in 118 mM K+, secretion did not increase further, suggesting that voltage changes are implicated in the control of the secretory process. 3. Blockade of secretion by increased concentrations of (+)-isradipine was much more manifest in polarized glands. The higher the degree of depolarization was (35, 59 or 118 mM K+), the lower the IC50 s were. So, the ratios between the IC50 s in polarized and depolarized glands rose from 3.92 in 35 mM K+ to 26.7 in 118 mM K+. 4. In contrast, the Ca2+ channel activator (-)-Bay K 8644 potentiated catecholamine release evoked by K+/Ca2+ pulses equally well in polarized or depolarized glands. The ratios between EC50 s in polarized or depolarized glands were, respectively, 0.30, 0.59 and 0.69 for 17.7, 35 and 118 mM K+. 5. In simultaneous experiments, the two enantiomers of Bay K 8644 exhibited opposite effects on secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Congener specific effects by polychlorinated biphenyls on catecholamine content and release in chromaffin cells

The effects of the non-planar polychlorinated biphenyl (PCB) congener 2,2′,4,4′-tetrachlorobiphenyl (2,4-TCB) and of the coplanar PCB congener 3,3′,4,4′-tetrachlorobiphenyl (3,4-TCB) were investigated on the catecholamine content and release from bovine adrenal chromaffin cells in culture. Each congener was tested at three concentrations (20, 50 and 100 μM) and two exposure periods (24 h and 5 days). Catecholamine release induced by K⁺-stimulation as well as catecholamine content of Triton X-100 treated cell cultures were examined using high-performance liquid chromatography (HPLC). 2,4-TCB showed dose- and time-dependent effects. 2,4-TCB at 100 μM reduced the K⁺-stimulated catecholamine release after 24 h of exposure. After 5 days of exposure, 2,4 TCB at 50 and 100 μM drastically reduced the K⁺-stimulated catecholamine release. 3,4-TCB even at a concentration of 100 μM over exposure of either 24 h or 5 days had no effects on the K⁺-stimulated secretion. When chromaffin cells, exposed to 2,4-TCB, were lysed with 0.5% Triton X-100, a dose- and time-dependent reduction of the catecholamine content appeared. The 3,4-TCB did not reduce the catecholamine content. Conversely there seemed to be a trend towards an increase in catecholamine content. Spontaneous release of catecholamines was strongly increased by the non-planar 2,4 TCB, while the coplanar 3,4 TCB showed no effects on this parameter. Furthermore, the effects of 2,4 TCB appeared to be reversible after replacing the highest concentration (100 μM) of the TCB-solution with culture-medium at the end of the 24-h exposure. Thus, K⁺-stimulated catecholamine release and the catecholamine content of bovine adrenal chromaffin cells was effectively reduced by the non-planar PCB congener whereas spontaneous catecholamine release was strongly increased. The coplanar PCB congener was ineffective at the same conditions. Received: 7 January 1997 / Accepted: 11 February 1997     

Effects of prostaglandin E1 on adrenal medulla chromaffin cells: model for studies of catecholamine release

The effects of prostaglandin E1(PGE1) on adrenergic mechanisms was studied, using adrenal medulla as a model tissue. Experiments were performed on male albino rats. PGE1 was introduced through the abdominal aorta. The adrenal medulla in the control and experimental animals was dissected and prepared for electronmicroscopic examinations. Catecholamine-containing vesicles in the chromaffin cells were counted and their size estimated. Statistical evaluation of the data showed that PGE1 caused an increase in the size of secretory vesicles and decrease in the total number of full vesicles, the effect being more pronounced on the adrenaline cells as compared to the noradrenaline cells.     

Characterization of ginseng saponin ginsenoside-Rg(3) inhibition of catecholamine secretion in bovine adrenal chromaffin cells.

Since ginsenoside-Rg(3), one of the panaxadiol saponins isolated from the ginseng root, significantly inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh), the properties of ginsenoside-Rg(3) inhibition were investigated. Although ginsenoside-Rg(3) inhibited the secretion evoked by ACh in a concentration-dependent manner, it affected the secretion stimulated by high K(+) or veratridine, an activator of the voltage-sensitive Ca(2+) or Na(+) channels, only slightly. The ACh-induced Na(+) and Ca(2+) influxes into the cells were also reduced by ginsenoside-Rg(3). The inhibitory effect of this saponin on the secretion of catecholamines was not altered by increasing the external concentration of ACh or Ca(2+). The ACh-evoked secretion of catecholamines was completely restored in cells that were preincubated with 10 microM ginsenoside-Rg(3) and then incubated without the saponin, whereas secretion was not completely restored in cells that were preincubated with 30 microM of this compound. Above 30 microM ginsenoside-Rg(3) increased the fluorescence anisotropy of diphenylhexatriene in the cells. Furthermore, the inhibitory effect of ginsenoside-Rg(3) at 30 microM on the ACh-evoked secretion of catecholamines was dependent upon the preincubation time, but this was not the case at 10 microM. These results strongly suggest that ginsenoside-Rg(3) blocks the nicotinic ACh receptor-operated cation channels, inhibits Na(+) influx through the channels, and consequently reduces both Ca(2+) influx and catecholamine secretion in bovine adrenal chromaffin cells. In addition to this action, the ginsenoside at higher concentrations modulates the fluidity of the plasma membrane, which probably contributes to the observed reduction in the secretion of catecholamines.     

Effects of genistein and herbimycin, tyrosine kinase inhibitors, on catecholamine release in bovine adrenal chromaffin cells

1 The effects of genistein and herbimycin, tyrosine kinase inhibitors, on catecholamine (CA) release were examined in bovine adrenal chromaffin cells. 2 In intact cells, genistein (10-100 microm) and herbimycin (3-30 microm) inhibited CA release induced by acetylcholine (ACh; 100 microm) or the nicotinic receptor stimulant 1,1-dimethyl-4-phenyl-piperazinium (DMPP; 10 microm), but did not affect CA release induced by high K+ (40 mm). 3 Genistein and herbimycin inhibited (45)Ca2+ uptake induced by ACh (100 microm). 4 Neither genistein nor herbimycin affected [(3)H]nicotine binding with nicotinic receptors. 5 In beta-escin-permeabilized cells, neither genistein nor herbimycin affected CA release induced by Ca2+ (1 microm). 6 These results suggest that protein tyrosine kinase plays the facilitatory role in the regulation of CA release induced by nicotinic receptor stimulation in stimulus-secretion coupling of bovine adrenal chromaffin cells.     

In vitro interaction between bovine adrenal medullary cell membranes and chromaffin granules: Specific control by Ca2+

Summary The control of the in vitro interaction between bovine adrenal medullary plasma membernes and chromaffin granules by calcium has been studied. This interaction, which has previously been shown to result in the release of the soluble granular content, is a possible cell-free model for exocytosis. The plasma memberane-induced catecholamine release was stimulated when the [Ca²⁺] exceeded 2×10^–7M. A maximal release was reached at 10^–5M with a half maximal response around 10^–6M. Mg²⁺ was not able to stimulate the system in the absence of Ca²⁺. These data suggest a high specificity of the calcium controlled exocytotic mechanism and the absence of an antagonism by high concentrations (10^–5–10^–1M) of magnesium at the exocytotic site itself.     

The effects of candesartan and ramipril on adrenal catecholamine release in anaesthetized dogs

We have investigated the effects of the angiotensin II type 1 receptor antagonist candesartan, and the angiotensin II converting enzyme inhibitor ramipril, on catecholamine release from the anaesthetized dog's adrenal gland. These drugs were given systemically in low and high doses. The gland was stimulated electrically (0.5-12 Hz) and by angiotensin II infusion (40 ng/kg/min). Electrical stimulation resulted in frequency-dependent increases in catecholamine release. Candesartan (0.8, 4.0 mg/kg) and ramipril (0.125, 0.625 mg/kg) increased basal catecholamine release along with decreases in blood pressure. Both drugs diminished direct nerve stimulation-induced catecholamine release. When both drugs were combined, their inhibitory effect was slightly enhanced. Candesartan blocked catecholamine release induced by angiotensin II. Ramipril was not tested in this respect. The percentage of noradrenaline released during electrical stimulation of the gland remained constant and ranged from 14% to 22%. Both drugs appear to act by blocking local modulation of catecholamine release by the chromaffin cells.     

Effects of the glycine prodrug milacemide (2-N-pentylaminoacetamide) on catecholamine secretion from isolated adrenal medulla chromaffin cells.

1. Milacemide (2-n-pentylaminoacetamide) is a glycine prodrug which readily crosses the blood brain barrier and increases brain glycine and glycineamide. In vitro and in vivo studies, with numerous tissues, including adrenal chromaffin cells, have clearly shown that the formation of the latter metabolites is exclusively mediated by monoamine oxidase B for which milacemide is a substrate. 2. Milacemide, glycineamide and glycine caused a time- and dose-dependent release of catecholamines from bovine isolated chromaffin cells. 3. Milacemide (10(-4) M) induced catecholamine release was roughly 30% of that initiated by acetylcholine (10(-4) M), the natural secretagogue. 4. The combined effects of milacemide (10(-4) M) and acetycholine (10(-4) M) on catecholamine secretion from chromaffin cells is additive, suggesting that milacemide does not act through the normal nicotinic receptor release mechanism. 5. The release of catecholamines from chromaffin cells in response to milacemide (10(-4) M) was partially inhibited by the selective MAO-B inhibitors (-)-deprenyl (10(-7) M) and AGN 1135 (10(-6) M). This indicates that the MAO-B derived metabolites, glycineamide and glycine, contribute to the secretion of catecholamines as does milacemide itself. 6. It is apparent that release of catecholamines by glycine is mediated by its uptake into the cells since [3H]-glycine uptake and catecholamine release showed a highly significant correlation (r = 0.96).     

Blood pressure responses to adrenal field stimulation as a measure of adrenal catecholamine release.

1 A new method for studying adrenal medullary release is described in pithed rats using field stimulation of an entire adrenal gland. 2 The increases in blood pressure in response to field stimulation of the gland consisted of an initial short, variable component, due to stimulation of adrenergic vasomotor neurones and a secondary, longer lasting component, due to medullary catecholamine release. 3 Removal of the initial component by drugs or cardiac/coeliac ganglionectomy did not affect the magnitude of the secondary pressor component. 4 The secondary pressor component was frequency-dependent, reproducible and stable with time. The extent of medullary catecholamine release could be assessed by comparing the blood pressure rise with those obtained after adrenaline injections. 5 The method appears to provide a reliable means by which adrenal medullary catecholamine release may be assessed without significant interference from vasomotor nerves.     

Evidence that prostaglandins activate calcium channels to enhance basal and stimulation-evoked catecholamine release from bovine adrenal chromaffin cells in culture

The effects of prostaglandins (PGs) on catecholamine (CA) secretion and Ca2+ fluxes were studied in a primary culture of bovine chromaffin cells. PGD2, PGF2 alpha and PGE2 induced CA release from cultured bovine chromaffin cells in a concentration dependent manner (0.03-3 microM). PGD2, PGF2 alpha and PGE2 at 3 microM elicited maximum CA release of 0.043 +/- 0.001, 0.059 +/- 0.008, 0.062 +/- 0.002 micrograms/10(6) cells, respectively. Three micromolar of PGD2, PGF2 alpha and PGE2 enhanced CA release induced by acetylcholine (ACh) in a degree of 186 +/- 10, 206 +/- 6, 150 +/- 4% of control respectively. PGs also enhanced CA release induced by 20 mM K+, veratridine and A23187. In Ca2+-free medium, PGs failed to affect basal and caffeine (50 mM)-induced CA release. PGF2 alpha increased 45Ca uptake and showed additive effect with ACh on 45Ca uptake. Nicardipine (0.1-10 microM) suppressed CA release and 45Ca uptake induced by PGF2 alpha, while diltiazem and verapamil failed to affect these responses to PGF2 alpha. BAY K 8644 (1 microM) potentiated CA release and 45Ca uptake evoked by PGF2 alpha. These results suggest that PGs enhance basal and stimulation-evoked CA release from chromaffin cells possibly through facilitation of Ca2+ influx. The mechanisms of action of PGs in adrenal medulla are discussed.