The gadolinium ion: a potent blocker of calcium channels and catecholamine release from cultured chromaffin cells

Gadolinium (Gd³⁺) is a trivalent ion of the lanthanide series which has a high charge density and a similar ionic radius to Ca²⁺. The effects of gadolinium ions on [³H]noradrenaline output and ⁴⁵Ca fluxes during resting conditions and stimulation were investigated in cultured bovine chromaffin cells. Chromaffin cells isolated from bovine adrenal medullae were plated on collagen-coated dishes (10⁶ cells/dish) in a medium containing 10% fetal calf serum. Seven-day-old cells were used in all studies.Exposure of chromaffin cells to 0.05mM Gd³⁺ produced 80 ± 5% and 81 ± 4% inhibition of the secretory responses to 10^?4M acetylcholine and 56 mM K⁺ respectively. Doubling of the extracellular concentration of Gd³⁺ produced an 87 ± 3% and 100 ± 2% inhibition of the acetylcholine-induced and K⁺-induced release of [ ³H]noradrenaline. Gd³⁺ (0.05 mM) also produced a parallel shift to the right in the dose-response relationship between extracellular Ca²⁺ concentrations and [ ³H]noradrenaline output during acetylcholine stimulation. This observation suggests a competitive antagonism between Ca²⁺ and Gd³⁺ in stimulus-secretion coupling.Gd³⁺ (0.05 mM) was an effective inhibitor (92.3 ± 0.7%) of the uptake of ⁴⁵Ca into chromaffin cells induced by a depolarizing concentration (56 mM) of K⁺. Furthermore, Gd³⁺ also inhibited the increase in both Ca²⁺ ? Ca²⁺ exchange mechanism and [ ³H]noradrenaline output observed in chromaffin cells upon the reintroduction of Ca²⁺ into a Ca²⁺]-free incubation medium.The results, which were obtained using low concentrations of Gd³⁺, indicate that gadolinium is a powerful inhibitor of the Ca²⁺ movements which are required for triggering amine release from chromaffin cells by different secretagogues. Moreover, the results also suggest that Gd³⁺ might be a useful tool for release and electrophysiological studies in other Ca²⁺-dependent systems.     

Characterization of catecholamine release from deer adrenal medullary chromaffin cells

Isolated adrenal medullary chromaffin cells maintained in culture have been widely used to study neurosecretory events. Many of these studies have been conducted using cells obtained from the bovine adrenal. In this study we have cultured chromaffin cells from an alternative large animal model, the deer, and have conducted the first characterization of secretion from this preparation. Cervine chromaffin cells, preloaded with [3H]noradrenalin, displayed a strong secretory response to the cholinergic agonist carbachol, with a maximal secretion of approximately 28% cell content over 15 min. This response was reproduced by nicotinic but not muscarinic agonists and was similarly inhibited by nicotinic but not muscarinic antagonists. Nicotine-evoked secretion measured over a 15 min time period was inhibited approximately 50% by the L-type Ca2+-channel antagonist nifedipine and approximately 20% by N-type (omega-conotoxin GVIA) or N, P/Q-type (omega-conotoxin MVIIC) antagonists. In contrast the response was unaffected by omega-agatoxin IVA, a P/Q-type antagonist. In addition to nicotinic receptor stimulation, activation of PACAP or histamine H1 receptors resulted in a concentration-dependent increase in secretion. PACAP was approximately two-fold more effective than histamine although both were weaker secretagogues than nicotine. In contrast, cervine chromaffin cells did not respond to angiotensin II or bradykinin, two agents known to stimulate secretion from bovine chromaffin cells. These data provide an initial characterization of the secretory response from cervine adrenal medullary chromaffin cells indicating that there are marked similarities but also potentially significant differences between them and their far more extensively described bovine counterparts.     

Nicotinic receptor-mediated intracellular calcium release in cultured bovine adrenal chromaffin cells

When cultured bovine adrenal chromaffin cells were stimulated by a nicotinic agonist, carbamylcholine (0.3 mM) or 1,1-dimethyl-4-phenylpiperazinium (50 microM), in the Ca2+-free medium containing 0.1 mM ethyleneglycoltetraacetic acid, intracellular free Ca2+ concentration ([Ca2+]i) rose from approximately 90 to 149 nM. High K+ (56 mM) and veratridine (50 microM) had no effect on the [Ca2+]i in Ca2+-free medium. The carbamylcholine-evoked rise in [Ca2+]i was blocked by hexamethonium (0.1 mM) but not by atropine (1 microM). Furthermore, the carbamylcholine-evoked rise in [Ca2+]i was inhibited by an intracellular Ca2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (10 microM) but not by a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (20 microM). These results show the existence of intracellular Ca2+ store sites, from which Ca2+ is released upon nicotinic receptor stimulation, in cultured adrenal chromaffin cells.     

The involvement of specific phospholipase C isozymes in catecholamine release from digitonin permeabilized bovine adrenal medullary chromaffin cells

The role of phospholipase C (PLC) in exocytosis has been investigated using digitonin permeabilized, [(3)H]noradrenaline ([(3)H]NA) loaded, bovine adrenal medullary chromaffin cells. The PLC inhibitor U-73122 caused a concentration-dependent suppression of Ca(2+)-evoked [(3)H]NA release but increased basal release (that occurring in the absence of Ca(2+)). Preincubation with antibodies against PLCgamma1 or PLCbeta3 (but not PLCdelta1, delta2, beta1 and beta2) also inhibited [(3)H]NA release evoked by Ca(2+) and increased basal release, indicating that only specific PLC isozymes are involved in these actions. Interestingly, PLCgamma1 (but not PLCbeta3) antibodies inhibited the ability of Ca(2+) to increase PLC activity in these permeabilized cells. These data therefore suggest that PLCgamma1 activity may have a specific role in regulating the exocytotic response from the adrenal chromaffin cell.     

Reinvestigation of the effect of orexin A on catecholamine release from adrenal chromaffin cells

Orexins have been shown to be implicated in the regulation of adrenal medulla functions. However, there are still inconsistent investigations on the effects of orexins on catecholamine release from chromaffin cells in varying species. In the present study, using the carbon-fiber amperometry, we investigated whether orexin A would stimulate catecholamine release from rat and mouse adrenal chromffin cells. Puff application of orexin A dose-dependently induced amperometric currents in the cultured rat chromaffin cells, which was completely blocked by the selective OX1R antagonist SB-334867 or by the removal of extracellular calcium. Likewise, in the mouse adrenal medulla slices, orexin A also induced catecholamine release mainly through the activation of OX1R. These results gain insight into our understanding of the pharmacological relevance of orexin system in modulating neuroendocrine functions.     

Pertussis toxin attenuates clonidine inhibition of catecholamine release in adrenal chromaffin cells

Characteristics of the inhibitory action of clonidine on catecholamine release in bovine adrenal chromaffin cells were investigated. Clonidine at 3 x 10(-5) M inhibited acetylcholine (ACh)-evoked release by about 50%, but not catecholamine release evoked by high K+. Another alpha 2-agonist alpha-methyladrenaline was ineffective at inhibiting ACh-evoked release. The inhibition by clonidine of ACh-evoked release was not reversed by alpha 2-antagonists. Treatment of these cells with pertussis toxin reversed the inhibitory effect of clonidine, while it did not affect the inhibitory action of hexamethonium and of nifedipine. Therefore, clonidine inhibition of catecholamine release in these cells seems not to be mediated by the alpha 2-adrenoceptor, but might be mediated by a specific receptor for clonidine.     

Electrical excitability of cultured adrenal chromaffin cells.

1. Adult human and gerbil adrenal medullary cells were maintained in dissociated cell culture and studied by micro-electrode penetration. 2. In the best recordings, chromaffin cell transmembrane potentials exceeded -50mV. 3. Chromaffin cells were capable of generating all-or-nothing over-shooting action potentials, similar to those generated by sympathetic neurones. 4. The action potentials were blocked by tetrodotoxin (TTX, 10(-6)g/ml.) but were not blocked by removal of Ca or by CoCl2 (10 mM). We conclude that the action potentials are probably generated by a Na mechanism. 5. Chromaffin cells are depolarized by the iontophoretic application of acetylcholine (ACh). This depolarization was accompanied by an increased membrane conductance and could trigger action potentials. 6. Action potentials were also found in cells in fresh slices of gerbil adrenal medullae.     

Inhibitory effect of polymyxin B on catecholamine secretion from cultured bovine adrenal medullary cells

Incubation of cultured bovine adrenal medullary cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), was associated with increased secretion of catecholamine (CA) from the cells. Polymyxin B (PMB, 30-300 microM), a preferential inhibitor of protein kinase C, inhibited the TPA-induced secretion of CA. PMB also inhibited CA secretion induced by other secretagogues, the Ca2+ ionophore ionomycin (10 microM), 56 mM K+ or acetylcholine (ACh). Ionomycin, 56 mM K+ or ACh increased the concentration of intracellular free Ca2+ ([Ca2+]i) (measured using the fluorescent calcium indicator quin2), whereas TPA did not increase [Ca2+]i. PMB blocked the increase in [Ca2+]i induced by 56 mM K+ or ACh at concentrations similar to those inhibiting the secretion of CA. In contrast, PMB did not affect ionomycin-induced increase in [Ca2+]i. These results strongly suggest that CA secretion induced by TPA or ionomycin is mediated via activation of protein kinase C. The results further indicate that in 56 mM K+- or ACh-evoked CA secretion, PMB inhibits the secretion by blocking Ca2+ influx into the cells.     

Mechanism through which GABAA receptor modulates catecholamine secretion from bovine chromaffin cells.

The actions and mechanism of GABAergic modulation of catecholamine secretion from isolated bovine chromaffin cells were investigated. The GABAA receptor agonist muscimol induced a fast rise in cytosolic [Ca2+]. The mean peak increase was 290 +/- 30 nM over basal levels. The increase in cytosolic [Ca2+] was abolished in the absence of extracellular [Ca2+] and was blocked by the GABAA antagonist bicuculline and the dihydropiridine nifedipine. Muscimol also elicited the release of catecholamines and increased the bisoxonol fluorescence indicating a cell depolarization. The [Ca2+] entry was well correlated with muscimol-evoked catecholamine secretion. When cells were treated with muscimol and a second secretagogue, a biphasic behavior was revealed. Muscimol enhanced the catecholamine release evoked by low concentrations of nicotine or K+, whereas release obtained at high concentrations of nicotine or K+ was actually inhibited. When the muscimol effect on membrane potential was studied in the presence of low K+ or nicotine concentrations, an enhancement of the bisoxonol fluorescence was observed. This effect was reversed at high concentrations of both K+ and nicotine. Measurement of 36Cl- fluxes showed an increase in membrane permeability to Cl- during muscimol stimulation. The influx or efflux in Cl- was dependent on membrane potential. In normal conditions, with a K+ concentration of 5.4 mM, a Cl- efflux was observed by both radiometric techniques, with 36Cl- and by the use of the chloride-sensitive fluorescent probe 6-methoxy-N-(3-sulphopropil)quinolinium, as indicator of intracellular Cl-. At high nicotine (20 mM) or K+ concentrations (105 mM) a Cl- influx was observed using 6-methoxy-N-(3-sulphopropil)quinolinium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of a high affinity uptake system for catecholamines in cultured bovine adrenal chromaffin cells

The uptake of catecholamines was investigated in bovine adrenal chromaffin cells cultured for 2 and 7 days. These cells, after their attachment onto the collagen-coated plates, began to develop processes which progressively increased in length with time in culture. Process outgrowth of a few cells was apparent on day 2 in culture. However, by day 7 most of the chromaffin cells possessed processes with a mean length of52.68 ± 1.27μm(n= 202). There was found to exist in both 2-day and 7-day-old cultures an uptake mechanism for (?)noradrenaline which in many aspects simulated the neuronal uptake₁ transport system in that it was saturable, followed Michaelis-Menten kinetics, had a high affinity for (?)noradrenaline with apparentK_m values ranging from 0.35–0.48 μm (n = 4) and 0.46 ? 0.67 μm (n = 4) on day 2 and day 7 respectively. This uptake process was blocked by low concentrations of desipramine (10^?7 M), a specific inhibitor of uptake₁, and like the neuronal uptake mechanism exhibited absolute Na⁺ dependency in which Li⁺ could not adequately replace Na⁺. However, uptake by the chromaffin cells did not show any stereochemical specificity towards the (?) form of noradrenaline nor did it exhibit structural specificity for (?)noradrenaline over (?)adrenaline.Therefore, it appeared that a high affinity accumulating mechanism for noradrenaline was present in cultured chromaffin cells isolated from adult bovine adrenal medulla. The properties of this uptake system did not vary significantly from day 2 to day 7 and thus did not parallel the morphological changes seen in culture.     

Microinjection of calmodulin antibodies into cultured chromaffin cells blocks catecholamine release in response to stimulation

Polyclonal monospecific antibodies raised in sheep against rat testis calmodulin demonstrated cross-reactivity with bovine adrenal medullary chromaffin cell calmodulin. This antibody immunoprecipitated a [35S]methionine-labelled protein from chromaffin cell extracts prepared from [35S]methionine prelabelled cells that comigrated on a sodium dodecylsulfate gel electrophoresis system with calmodulin. In addition, an excess of non-radioactive exogenous calmodulin was shown to readily compete with this labelled endogenous protein for the antibodies' binding sites. Erythrocyte ghosts were used as vehicles for microinjecting either preimmune immunoglobulin G or anti-calmodulin immunoglobulin G into chromaffin cells following a polyethylene glycol-induced cell fusion procedure. The efficiency of ghost cell fusion was monitored and found to be 43.6 +/- 1% (n = 33). Cell morbidity subsequent to fusion and microinjection was negligible (87.8 +/- 0.6% of the total cell population were viable cells; n = 33) as determined by the Trypan Blue exclusion test. The delivery of intact antibodies raised against calmodulin directly into the cytoplasm of cultured chromaffin cells by erythrocyte ghost-mediated microinjection, inhibited catecholamine output in response to stimulation by either acetylcholine (10(-4) M) or a depolarizing concentration of potassium (56 mM). However, under these conditions, the chromaffin cell's ability to accumulate exogenous catecholamines through a high affinity uptake system, as well as the kinetic parameters that characterize this uptake mechanism remained unaltered. Furthermore, microinjection of preimmune immunoglobulin G did not modify either catecholamine uptake or stimulation-induced amine release from chromaffin cells. It therefore appears that calmodulin may play a role in the process of stimulus-secretion coupling in the chromaffin cell in culture while it is of little significance to the high affinity amine uptake mechanism.     

Hypophosphorylated neurofilament subunits in the cytoskeletal and soluble fractions of cultured bovine adrenal chromaffin cells

The neurofilament proteins in cultured bovine adrenal chromaffin cells are in a hypophosphorylated state, as determined by the co-migration of the 160,000 and 210,000 molecular weight subunits with in vitro dephosphorylated bovine brain subunits on sodium dodecyl sulfate polyacrylamide gels. In addition, chromaffin cells were not stained by anti-heavy neurofilament subunit that binds only to phosphorylated epitopes. Pulse-labeling with 32Pi in the presence and absence of the protein synthesis inhibitor emetine indicated that some neurofilament protein phosphorylation occurred co-translationally and/or immediately after synthesis of the proteins. Pulse-chase experiments showed that the three neurofilament proteins rapidly attained their maximal phosphorylation levels, as multiple forms of either of the respective subunits were not seen after a one hour chase. We found that Triton X-100-soluble forms of high molecular weight neurofilament and middle molecular weight neurofilament subunits were present in chromaffin cells, and they also co-migrated with standard neurofilament proteins dephosphorylated in vitro. However, there were differences between the phosphopeptide maps of cytoskeleton-associated and soluble middle molecular weight neurofilament subunit, suggesting that the localization of phosphate moieties rather than extent of phosphorylation influences the association of the subunit with neurofilaments. Double immunofluorescence staining of cell cultures with antibody to the 70,000 molecular weight subunit and with anti-vimentin showed that chromaffin cells do not express vimentin.     

Catecholamine release from bovine chromaffin cells: the role of sodium-calcium exchange in ouabain-evoked release

Spontaneous catecholamine (CA) release from bovine chromaffin cells maintained in primary tissue culture has been measured after pre-loading the cells with [3H]noradrenaline. Ouabain inhibited 86Rb+ uptake and increased 3H release in a concentration-dependent manner during a 60 min incubation period. Low external Na+ (5 mM: Li+ substitution) also increased 3H release. Whereas the 3H-releasing action of ouabain was maintained, the Li(+)-evoked release decreased with time. The effects of both ouabain and low Na+ solution on 3H release were completely inhibited by removal of Ca2+ from the external medium even though in Ca2(+)-free solution ouabain further inhibited 86Rb+ uptake into the cells. Readmission of Ca2+ to Na(+)-loaded cells (10-4 M-ouabain in Ca2(+)-free-1 mM-EGTA solution for 60 min) markedly increased the release of 3H. In the additional presence of diphenylhydantoin (DPH, 10-4 M) 3H release was significantly less on Ca2+ readmission. The 3H release from Na(+)-loaded cells was proportional to the concentration of Ca2+ readmitted. The 3H release was further increased from Na(+)-loaded cells in response to Ca2+ readmission when [Na+]o was lowered from 149 to 5 mM (Li+, choline+, Tris+ or sucrose substitution) though Li+ was less effective than the other Na+ substitutes. Potassium removal from the external medium significantly inhibited the 3H release evoked by Ca2+ readmission to Na(+)-loaded cells, even when [Ca2+]o was greater than normal (7.5 mM) or if Ca2+ was readmitted in low [Na+]o solution. Rb+, Cs+ or Li+ could substitute for K+ with the order of potency: Rb+ greater than or equal to K+ greater than Cs+ greater than Li+. A slight increase of external K+ (10.8 mM) potentiated the 3H release from Na(+)-loaded cells on Ca2+ readmission, but a higher concentration of K+ (149.4 mM) had the opposite action. The data is consistent with the hypothesis that ouabain-evoked CA release from bovine chromaffin cells is, in part, a consequence of an internal Na(+)-dependent Ca2+ influx. The evidence also suggests that there is Na(+)-Ca2+ competition at the external arm of the exchanger together with a monovalent cation activation site.     

Effects of pro-opiomelanocortin fragments on release of catecholamines from adrenal chromaffin cells

The effects of pro-opiomelanocortin (POMC) peptide fragments on the basal and agonist-induced release of catecholamines (CAs) from monolayer cultures of purified bovine adrenal chromaffin cells were tested. None of the 5 peptides tested, i.e. ß-MSH, ACTH_1–39, γ-MSH_1–13. γ₃-MSH and N-terminal POMC fragment, had any effect on basal CA release. However, ß-MSH (10^?5 M), γ-MSH_1–13 (10^?6–10^?5 M), γ₃-MSH (10^?5 M) and N-terminal POMC fragment (10^?5 M) inhibited the nicotine-induced release of CAs from the chromaffin cells. The possible physiological significance of this inhibitory neuromodulation is discussed.     

Mechanism of histamine-induced calcium efflux from cultured bovine adrenal chromaffin cells: possible involvement of an Na/Ca exchange mechanism

The effect of stimulation of the histamine receptor on Ca²⁺ mobilization in cultured bovine adrenal chromaffin cells was examined. Histamine (10⁻⁵ M) increased the intracellular free Ca²⁺ ([Ca²⁺]_i) to a peak in the presence or absence of extracellular Ca²⁺, followed by decrease with time. Histamine (10⁻⁸–10⁻⁵ M) also stimulated ⁴⁵Ca²⁺ efflux from cultured bovine adrenal chromaffin cells in a concentration dependent manner. Its stimulatory effect on ⁴⁵Ca²⁺ efflux was inhibited by the specific histamine H₁ receptor antagonist mepyramine. The increase in histamine-stimulated ⁴⁵Ca²⁺ efflux was inhibited by deprivation of extracellular Na⁺ and by the Na⁺/Ca²⁺ exchange inhibitor amiloride. In addition, histamine stimulated ²²Na⁺ influx into the cells, and this action was inhibited by amiloride. These results suggest that stimulation of the histamine H₁ receptor regulates Na⁺/Ca²⁺ exchange in cultured bovine adrenal chromaffin cells.     

Stimulatory effect of vasoactive intestinal polypeptide on catecholamine secretion from isolated guinea pig adrenal chromaffin cells

The stimulatory effect of vasoactive intestinal polypeptide (VIP) on catecholamine (CA) secretion from isolated guinea pig adrenal chromaffin cell was studied. VIP (1-10 microM) induced dose-dependent CA secretion, which was slow and continued for at least 30 min. This VIP-induced CA secretion was dependent on the presence of Ca2+ in the medium, but no significant increase in Ca2+ uptake by the cells was observed during their stimulation with VIP. Studies on the intracellular free Ca2+ level ([Ca2+]i) using fura-2 showed that acetylcholine and muscarine induced a marked increase in the [Ca2+]i, but that VIP induced only a slight increase. Thus VIP may induce CA secretion by increasing the sensitivity of the secretion of CA to Ca2+.     

Swelling-induced catecholamine secretion recorded from single chromaffin cells

We have studied osmotically induced catecholamine secretion from bovine adrenal chromaffin cells by combining patch-clamp measurements, electrochemical detection of secretion, and Fura-2 measurements of intracellular free calcium concentration ([Ca²⁺]_i). We find that osmotically induced catecholamine release is exocytotic and calcium dependent. Furthermore, we demonstrate that cell swelling is coupled to such secretion via a volume-activated current, carrying predominantly chloride, which causes a plateau depolarization of the cell membrane potential and thus promotes voltage-activated calcium influx. Therefore, cell volume changes may modulate the secretory activity.     

Transplantation of cultured fetal human adrenal chromaffin cells to rat brain

Adrenal chromaffin cells, obtained from a therapeutically aborted human fetus of about 11 weeks gestation, were cultured for 3 weeks in vitro and then transplanted to the striatum of rats. Transplanted cells became established through strands of tissue growing into the host striatum. No signs of inflammation or rejection were observed up to the time of sacrifice one month post-transplantation. Histofluorescence examination of the implanted areas showed many clusters of cells having an intensely positive catecholamine fluorescence with some of the cells developing conspicuous processes. This study, showing survival of cultured human adrenal chromaffin cells transplanted into rat brain tissue, might indicate the feasibility of using cultured human material for future human neuronal transplantation studies as a therapeutic measure.     

Nitric oxide modulates evoked catecholamine release from canine adrenal medulla.

Nitric oxide has various actions, acting in a neurotransmitter-like role and also as a paracrine messenger between vascular endothelial and smooth muscle cells. This study was done to determine whether endogenous nitric oxide has a role in modulating evoked catecholamine release from the canine adrenal medulla. Isolated adrenal glands were perfused with Krebs-Ringer solution as a control, or with Krebs-Ringer solution containing either N(G)-monomethyl-L-arginine (L-NMMA; 3x10(-4) M) to non-selectively inhibit nitric oxide synthase or 7-nitroindazole (10(-4) M), a relatively selective inhibitor of neuronal nitric oxide synthase. Catecholamine release was evoked using the nicotinic cholinergic agonist 1,1-dimethyl-4-phenylpiperazinium iodine. From the collected perfusate epinephrine, norepinephrine, and dopamine were measured by high performance liquid chromatography. Previous studies have shown that in the presence of L-NMMA, basal releases of epinephrine, norepinephrine and dopamine are increased. 7-Nitroindazole had no effect on basal catecholamine release, suggesting that nitric oxide from an endothelial source was responsible for the inhibition of basal catecholamine release from the adrenal medulla. Epinephrine and norepinephrine releases were augmented when either of the nitric oxide synthase inhibitors was added during submaximal nicotinic stimulation, indicating that endogenous nitric oxide inhibited release of epinephrine and norepinephrine. Both neuronal and endothelial nitric oxide synthases appeared to be responsible for this inhibition.In summary, these studies suggest that nitric oxide, from both neuronal and endothelial sources, modulates evoked catecholamine release from canine adrenal medulla, while nitric oxide from an endothelial source is most likely responsible for modulation of catecholamine release under basal conditions.