Intact Bovine Adrenal Chromaffin Cells Research Articles

Surface and intracellular nicotinic receptors expressed in intact adrenal chromaffin cells: direct measurements using [ 3H]epibatidine

The presence and importance of assembled, intracellular neuronal nicotinic acetylcholine receptors (nAChRs) has not been established in native systems. In these studies [ 3H]epibatidine binding techniques were used to characterize surface and intracellular sites expressed in intact bovine adrenal chromaffin cells in culture. Permeant (300 μM nicotine) and impermeant (5 mM carbachol) cholinergic agents were used to define specific [ 3H]epibatidine binding to total (surface and intracellular) sites and surface sites, respectively. Intracellular [ 3H]epibatidine binding sites were characterized after eliminating surface binding sites via alkylation. Equilibrium binding to all sites was reached within 30 min at room temperature. Homologous (epibatidine) competition experiments on total (surface and intracellular) binding sites demonstrated a significant fraction of the high affinity sites were localized to intracellular compartments. Saturation binding assays to surface and intracellular sites revealed K d values of 1.9±1.1 and 3.6±1.9 nM, respectively. These binding studies document the existence of a significant population of high affinity, intracellular binding sites in native neuronal cells and support their characterization as assembled, α3β4* nAChRs. Although the intracellular nAChRs represent ∼70% of the total, high-affinity nAChRs expressed in cultured chromaffin cells, they do not appear to be involved in functional recovery after nAChR down-regulation.

Simultaneous measurement of tyrosine hydroxylase activity and phosphorylation in bovine adrenal chromaffin cells

A method for simultaneous measurement of tyrosine hydroxylase (TH) activation and phosphorylation in permeabilised and intact bovine adrenal chromaffin cells (BACCs) was established. Permeabilised cells were stimulated with cyclic AMP (1–10 μM) in the presence of [ 32P]ATP and l-[carboxyl- 14C]tyrosine. Intact BACCs were preincubated with 32P i for 3 h and stimulated with forskolin (1–5 μM) in the presence of l-[carboxyl- 14C]tyrosine. On stimulation each well was covered with a sealed ‘chimney’ fitted with a small plastic cup containing 300 μl of 1.0 M NaOH that trapped the 14CO 2 released. TH activity was determined by measuring 14C radioactivity. TH phosphorylation was measured in the same cells by separating the solubilized proteins on SDS PAGE followed by autoradiography and/or HPLC analysis. It was found that H89, a protein kinase A inhibitor, significantly blocked both TH phosphorylation and activation in response to cyclic AMP in permeabilised cells. However, in intact cells, H89 was effective only in respect to forskolin-stimulated TH activity and did not block the forskolin-stimulated TH phosphorylation of Ser-40. The reason(s) for this lack of correlation between TH activation and phosphorylation is presently not understood.

The interaction of tetanus toxin with intact bovine adrenal chromaffin cells: Binding of toxin and subsequent inhibition of catecholamine release

Tetanus toxin (about 1 nM) inhibits 70% of the nicotine-evoked release of catecholamines from intact adrenal medullary chromaffin cells after 20 h of incubation and 30% of the K +-evoked release. Inhibition of Ca 2+-evoked release from detergent-permeabilized cells requires higher concentrations of toxin (about 1 μM) toxin, but is maximal after 12 min. Preincubation of the intact cells with ganglioside GT1 in the absence of toxin also inhibits evoked secretion. 125I-labelled toxin bound specifically to these cells; the binding capacity was greater at pH 6 (about 1 pmol toxin/mg cell protein) than at pH 7.4 (about 0.25 pmol). In both cases there were at least two binding components: one of high affinity ( K d about 1 nM) accounting for about 20% of total binding and one of lower affinity ( K d 10–20 nM). Preincubation of the cells with ganglioside increased the binding capacity, but did not affect the K d of the lower affinity component. Similar observations could be made when binding was measured immunocytochemically. Extraction of gangliosides from chromaffin cells and overlay experiments with radiolabelled toxin showed that, as well as GM3, the major ganglioside component of chromaffin cell membranes, a ganglioside having the chromatographic mobility of GTI was a major ligand for toxin.

Specific binding of inositol hexakisphosphate (phytic acid) to adrenal chromaffin cell membranes and effects on calcium-dependent catecholamine release

In a membrane preparation of cultured bovine adrenal chromaffin cells, [ 3H]inositol hexakisphosphate ([ 3H]InsP 6) was shown to bind specifically with a k d of 90 nM and a B max of 700 fmol/mg protein. The Hill coefficient was not significantly different from unity. The association of [ 3H]InsP 6 was slow, with equilibrium binding being reached within 10 min. The dissociation of [ 3H]InsP 6 showed monophasic kinetics. In [ 3H]InsP 6 competition binding experiments, we found that reduction in the number of phosphorylated sites in inositol resulted in a gradual loss of binding potency. In intact bovine adrenal chromaffin cells, InsP 6 elicited a concentration-dependent facilitation of 45C 2+ influx along with the release of the catecholamines, epinephrine and norepinephrine. The latter responses were slower and longer-lasting than responses to depolarizing stimuli, such as nicotine and high K +. The catecholamine release required the presence of extracellular Ca 2+. In good agreement with the binding studies, lower inositol phosphates displayed reduced secretagogue potency. In conclusion, in bovine adrenal chromaffin cells, InsP 6 appeared to bind to specific sites and elicited Ca 2+ influx and catecholamine release.

A distinct 2,5-di-( tert-butyl)-1,4-benzohydroquinone-sensitive calcium store in bovine adrenal chromaffin cells

The fluorescent Ca 2+ indicator Fura 2 was used to monitor Ca 2+ release induced by the Ins(1,4,5)P 3-mobilizing agonist angiotensin II (Ag II), caffeine and 2,5-di-( tert-butyl)-1,4-benzohydroquinone (tuBHQ), in intact bovine adrenal chromaffin cells. Under low external Ca 2+ conditions, tuBHQ, Ag II and caffeine elicited Ca 2+ rises, indicating Ca 2+ release from internal stores. Prior addition of Ag II had no noticeable effect on the extent of release of Ca 2+ induced by tuBHQ. Stimulation of the cells with tuBHQ before either Ag II or caffeine, similarly had no effect on Ca 2+ released by these two agonists. It was concluded, therefore, that there is a third intracellular Ca 2+ store in bovine adrenal chromaffin cells, distinct and non-overlapping, from those sensitive to caffeine or Ins(1,4,5)P 3-Mobilizing agonists.

The inhibition by pertussis and tetanus toxins of evoked catecholamine release from intact and permeabilized bovine adrenal chromaffin cells

Pertussis toxin stimulates both basal and nicotine-evoked catecholamine secretion from intact bovine adrenal chromaffin cells, as well as Ca 2+-evoked release from permeabilized cells. Tetanus toxin inhibits all these effects; it reduces the secretion of intact cells treated with pertussis toxin to the basal level, and decreases by about 50% Ca 2+-evoked release from permeabilized cells whether or not previously stimulated by pertussis toxin.

Simultaneous measurements of cytosolic calcium and secretion in single bovine adrenal chromaffin cells by fluorescent imaging of fura-2 in cocultured cells.

The cytosolic free calcium concentration ([Ca2+]i) and exocytosis of chromaffin granules were measured simultaneously from single, intact bovine adrenal chromaffin cells using a novel technique involving fluorescent imaging of cocultured cells. Chromaffin cell [Ca2+]i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-loaded NIH-3T3t cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca2+ response to ATP, which is coreleased with catecholamine from the chromaffin cells. In response to the depolarizing stimulus nicotine (a potent secretagogue), chromaffin cell [Ca2+]i increased rapidly. At the peak of the response, [Ca2+]i was evenly distributed throughout the cell. This elevation in [Ca2+]i was followed by a secretory response which originated from the entire surface of the cell. In response to the inositol 1,4,5-trisphosphate (InsP3)-mobilizing agonist angiotensin II (a weak secretagogue), three different responses were observed. Approximately 30% of chromaffin cells showed no rise in [Ca2+]i and did not secrete. About 45% of the cells responded with a large (greater than 200 nM), transient elevation in [Ca2+]i and no detectable secretory response. The rise in [Ca2+]i was nonuniform, such that peak [Ca2+]i was often recorded only in one pole of the cell. And finally, approximately 25% of cells responded with a similar Ca2+-transient to that described above, but also gave a secretory response. In these cases secretion was polarized, being confined to the pole of the cell in which the rise in [Ca2+]i was greatest.(ABSTRACT TRUNCATED AT 250 WORDS)

Internal Ca 2+ mobilization and secretion in bovine adrenal chromaffin cells

Since secretion from intact bovine adrenal chromaffin cells in response to depolarization by nicotine is triggered by a rise in the concentration of intracellular Ca 2+ ([Ca 2+] i) to about 200–300 nM above basal, it has been assumed that the failure of the inositol 1,4,5-trisphosphate (InsP 3)-mobilizing muscarinic agonists to induce secretion reflects the fact that the 50 nM rise in [Ca 2+] i they elicit is insufficient to trigger the exocytotic machinery. A recent report [11], however, has demonstrated that some of the nicotine-induced rise in [Ca 2+] i could originate from the InsP 3-releasable Ca 2+ store. The role of this Ca 2+ store in secretion from bovine adrenal chromaffin cells is therefore unclear. In order to investigate in more detail the role of the InsP 3-sensitive Ca 2+ store in secretion from these cells, we have used a combination of an InsP 3-mobilizing muscarinic agonist and the sesquiterpene lactone thapsigargin (TG), which releases internal Ca 2+ without concomitant breakdown of inositol lipids or protein kinase C activation, to examine the events which follow depletion of the releasable Ca 2+ store in these cells. Monitoring [Ca 2+] i using Fura-2 demonstrated that TG released Ca 2+ from the InsP 3-sensitive store and, additionally, that the Ca 2+ response to TG was composed of two distinct, temporally separated, components: a) a slow (1 min) increase in [Ca 2+] i to ∼50 nM above basal that was independent of extracellular Ca 2+ and b) the maintenance of this level at a new steady-state that was dependent on the continual entry of extracellular Ca 2+. Since the entry of external Ca 2+ always followed on from the release of stored Ca 2+, it is conceivable that Ca 2+ flows into the cell via the store after it has first been depleted by TG. A link similar to that proposed by Putney [28] may therefore exist between the Ca 2+ store and the external milieu in these cells. The results also showed that, despite raising [Ca 2+] i by only 50 nM, TG was able to elicit a secretory response. This result demonstrates that it is possible to induce secretion from these cells by a modest elevation in [Ca 2+] i resulting from internal Ca 2+ release, provided that mobilization is followed by Ca 2+ influx. These results, therefore, argue against the lack of efficacy of muscarinic agonists being a result of their inability to sufficiently elevate [Ca 2+] i, and point towards Ca 2+ influx distal to internal release as important for secretion due to some stimuli.

Binding of prostaglandin E 2 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) E 1 and E 2. We have now investigated the binding of PGE 2 to intact bovine adrenal chromaffin cells and the effects of prostaglandins on the release of catecholamines from these cells. Adrenal chromaffin cells specifically bound PGE 2 with a dissociation constant of 2 nM and a concentration of about 40 000 binding sites per cell. Low concentrations of PGE 2 inhibited the nicotine-stimulated release of catecholamines from these cells. The effect of PGE 2 was biphasic, the maximal inhibitory effect being observed at a concentration of between 1 and 10 nM. Higher concentrations (1 μM) of PGE 2 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 PGE 2 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 PGE 2 plays a modulatory role in the regulation of catecholamine secretion from the adrenal medulla.

Oligopeptide inhibitors of metalloendoprotease activity inhibit catecholamine secretion from bovine adrenal chromaffin cells by modulating intracellular calcium homeostasis.

Synthetic oligopeptide inhibitors of metalloendoprotease activity were found to inhibit catecholamine release from intact bovine adrenal chromaffin cells. The efficiency of these compounds in blocking secretion was dependent on the type and dose of the secretagogues employed. By contrast, catecholamine release from digitonin-permeabilized cells stimulated with micromolar calcium was virtually not affected. Using a different model system mimicking protein-mediated membrane fusion during exocytosis (Bental, M., Lelkes, P.I., Scholma, J., Hoekstra, D., and Wilschut, J. (1984) Biochim. Biophys. Acta 774, 296-300) we found that exposure of chromaffin granules to a genuine metalloendoprotease, thermolysin, impaired their fusion competence with liposomes. The same oligopeptide inhibitors of metalloendoprotease activity that interfered with secretion from the intact cells were also found to cause an increase in 45Ca2+ efflux concomitant with a slight elevation of the free intracellular calcium concentration [( Ca2+]i) to levels not sufficient to elicit secretion. Subsequent stimulation of the cells in the presence of the potent inhibitors resulted in a reduced increase in the cytosolic calcium concentration, as compared to nontreated control cells. The reduction in the secretagogue-evoked rise in [Ca2+]i was also dependent on the time of pretreatment of the cells with the metalloendoprotease inhibitors. Consistently, none of these effects were seen with structurally similar oligopeptides that are not metalloendoprotease substrates/inhibitors. We conclude that potent inhibitors of metalloendoprotease activity and hence, presumably, the enzymes per se modulate stimulus-secretion coupling by interfering with calcium homeostasis rather than directly with membrane fusion.