Single Bovine Adrenal Chromaffin Cells Research Articles

Generation of functionally competent single bovine adrenal chromaffin cells from cell aggregates using the neutral protease dispase

A simple and efficient procedure has been developed to enzymatically dissociate aggregates of bovine adrenal chromaffin cells in suspension culture into viable, responsive single cells. For dissociation, the neutral protease dispase is added directly to the culture medium for a minimum of 3 h, followed by incubation of the cells in Hank’s calcium–magnesium-free balanced salt solution at 37 °C with intermittent trituration to facilitate dispersion. This procedure generates a population of phase-bright single cells that are round in morphology, take up the dye neutral red, exclude the dye trypan blue and readily attach to tissue culture dishes coated with collagen, fibronectin or polylysine, thereby permitting applications that require plated-down conditions. When transferred to culture medium, the cells begin to reaggregate. By altering the length of time the cells are incubated in culture medium prior to attachment, the degree of reaggregation can be controlled to obtain plate-down profiles that consist of both isolated cells and cells in aggregates of varying sizes. Returning dissociated cells to suspension culture results in the reformation of large cell aggregates. Several measures of chromaffin cell function were indistinguishable for dissociated cells placed either in monolayer culture or suspension culture versus non-dissociated cells, implying that the dissociation procedure does not alter cellular responses or cause cellular damage.

The Sea Anemone Toxin Bc2 Induces Continuous or Transient Exocytosis, in the Presence of Sustained Levels of High Cytosolic Ca2+ in Chromaffin Cells

We have isolated and characterized a new excitatory toxin from the venom of the sea anemone Bunodosoma caissarum, named Bc2. We investigated the mechanism of action of the toxin on Ca(2+)-regulated exocytosis in single bovine adrenal chromaffin cells, monitoring simultaneously fura-2 fluorescence measurements and electrochemical recordings using a carbon fiber microelectrode. Bc2 induced quantal release of catecholamines in a calcium-dependent manner. This release was associated with a sustained rise in cytosolic Ca(2+) and displayed two different patterns of response: a continuous discharge of prolonged duration that changed to a transient burst as the toxin concentration (or incubation time) increased. Continuous secretion was dependent on the activity of native voltage-dependent Ca(2+) channels and showed a pattern similar to that of alpha-latrotoxin; however, its kinetics adjusted better to that of continuous cell depolarization with high K(+) concentration. In contrast, transient secretion was independent of Ca(2+) entry through native voltage-dependent Ca(2+) channels and showed inhibition of late vesicle fusion that was accompanied by "freezing" of F-actin disassembly. These new features make Bc2 a promising new tool for studying the machinery of neurotransmitter release.

Direct Visualization of Secretion from Single Bovine Adrenal Chromaffin Cells by Laser-Induced Native Fluorescence Imaging Microscopy

Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved with laser-induced native fluorescence imaging microscopy. By monitoring the native fluorescence of catecholamines excited by the 275 nm laser line with an intensified charge-coupled-device (CCD) camera, we obtained good temporal and spatial resolution simultaneously without using additional fluorescent probes. Large variations were found among individual cells in terms of the amounts of catecholamines secreted and the rates of secretion. Different regions of a cell also behave differently during the secretion process. However, the degree of this local heterogeneity is smaller than in neurons and neuralgia. The influence of deep-ultraviolet (UV) laser excitation on cells is also discussed. This quantitative imaging technique provides a useful noninvasive approach for the study of dynamic cellular changes and the understanding of the molecular mechanisms of secretory processes.

On-column monitoring of secretion of catecholamines from single bovine adrenal chromaffin cells by capillary electrophoresis

The secretion of catecholamines from individual bovine adrenal medullary cells was quantitatively monitored by capillary electrophoresis with laser-induced native fluorescence detection. By using a physiological balanced-salt solution as the running buffer for CE, the amount of norepinephrine (NE) and epinephrine (E) secreted by their physiological secretagogue, acetylcholine, and the amount remaining in a single cell can be simultaneously quantified. Among the six different glands (from separate cows) studied, a predominance of E-rich cells were found. There was no apparent relationship between the ratio of NE/E released and the original NE/E content in the cell. The secretion process was also monitored dynamically with this method by continuously passing acetylcholine over the cell during stimulation. From the peak width and shape of the released material, one can estimate the time scale of the release process.

Protein Kinase C Enhances Exocytosis from Chromaffin Cells by Increasing the Size of the Readily Releasable Pool of Secretory Granules

We have used membrane capacitance measurements to assay Ca 2+-triggered exocytosis in single bovine adrenal chromaffin cells. Brief application of phorbol ester (PMA) enhances depolarization-evoked exocytosis severalfold while actually decreasing the Ca 2+ current. Ca 2+ metabolism is unchanged. Three different protocols were used to show that PMA increases the size of the readily releasable pool of secretory granules. PMA treatment leads to a large increase in amplitude, but little change in the time course of the exocytic burst that results from rapid elevation of [Ca 2+] i upon photolysis of DM-Nitrophen. Thus, PKC appears to affect a late step in secretion but not the Ca 2+ sensitivity of the final step.

Role of [Na+]i and [Ca2+]i in nicotine-induced norepinephrine release from bovine adrenal chromaffin cells.

Intracellular free sodium ([Na+]i) and calcium ([Ca2+]i) concentrations were determined by sodium-binding benzofuran isophthalate (SBFI) and fura 2 microfluorimetry, respectively, in bovine adrenal chromaffin cells (BCC). Validation of SBFI microfluorimetry by in vitro and in vivo calibration revealed a reliable assessment of [Na+]i within a range of 1-30 mM in single BCC. Nicotine (0.1-10 microM) induced concentration-dependent increases of both [Na+]i (from 3.3 +/- 0.1 to 25.6 +/- 0.4 mM, n = 76, P < 0.001) and [Ca2+]i (from 64 +/- 1 to 467 +/- 16 nM, n = 87, P < 0.001), which were accompanied by an increase in [3H]norepinephrine (NE) release. Consistent with an exocytotic release mechanism, nicotine-induced increments of [Ca2+]i and [3H]NE release were reduced under calcium-free conditions and by gadolinium chloride (40 microM), whereas [Na+]i was not affected. In contrast, a parallel attenuation of nicotine-evoked changes in [Na+]i, [Ca2+]i, and [3H]NE release was observed during reduction of the extracellular sodium concentration. The nicotine-evoked responses were neutralized by the nicotinic receptor antagonist hexamethonium (100 microM) but not by blockade of voltage-dependent sodium channels (1 microM tetrodotoxin). In conclusion, the nicotine-induced exocytotic release of [3H]NE is triggered by an increase in [Ca2+]i, which is facilitated by sodium influx through the nicotinic receptor ionophore.

Spatial localization of agonist-induced Ca2+ entry in bovine adrenal chromaffin cells. Different patterns induced by histamine and angiotensin II, and relationship to catecholamine release.

The spatial organization of agonist-induced Ca2+ entry in single bovine adrenal chromaffin cells has been investigated using video-imaging techniques to visualize fura-2 quenching by the Ca2+ surrogate, Mn2+. The potent secretagogue histamine, in addition to releasing Ca2+ from intracellular stores, resulted in a large influx of external Mn2+ that occurred over the entire surface of the cell. The influx of Ca2+ that this mirrors was found to be an obligatory requirement for the triggering of catecholamine release by histamine, which suggests that such a global influx of Ca2+ into the cell probably underlies the ability of this agonist to stimulate a large secretory response. By contrast, the weaker secretagogue angiotensin II, which also acts through the second messenger inositol trisphosphate, produced a localized entry of external Mn2+ in 64% of cells. In these cells, localized Mn2+ entry always occurred at the pole of the cell in which the angiotensin II-induced rise in [Ca2+]i was largest. Since exocytosis in response to angiotensin II has previously been shown to be restricted to this same pole of the cell (Cheek et al. (1989). J. Cell Biol. 109, 1219-1227), these results suggest that localized influx of Ca2+ in response to angiotensin II could underlie the polarized exocytotic response observed with this stimulus. These results directly demonstrate that different agonists can induce different patterns of divalent cation influx in the same cells and, furthermore, suggest how these different patterns can have a direct influence on cellular function.

Ca2+ influx induced by the Ca(2+)-ATPase inhibitors 2,5-di-(t-butyl)-1,4-benzohydroquinone and thapsigargin in bovine adrenal chromaffin cells.

We have characterized the effect of the Ca(2+)-ATPase inhibitors 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ) and thapsigargin on the concentration of cytosolic Ca2+ in single bovine adrenal chromaffin cells by video-imaging of fura-2-loaded cells. Addition of either inhibitor released Ca2+ from internal stores in the absence of external Ca2+. tBHQ was unable to stimulate further Ca2+ release after addition of thapsigargin, but thapsigargin could do so after release by tBHQ, indicating that the tBHQ-sensitive stores are a sub-set of those sensitive to thapsigargin. Angiotensin II was able to elicit Ca2+ release after application of tBHQ, indicating that at least part of the tBHQ-sensitive stores were distinct from those discharged by Ins(1,4,5)P3. In the presence of external Ca2+, both Ca(2+)-ATPase inhibitors produced a more prolonged rise in cytosolic Ca2+ consistent with stimulated Ca2+ entry. The ability of the inhibitors to activate a Ca(2+)-entry pathway was confirmed by monitoring quenching of fura-2 after stimulated entry of the Ca2+ surrogate Mn2+. These findings indicate that bovine adrenal chromaffin cells possess a mechanism by which Ca2+ entry can be activated, following emptying of certain internal stores, independently of receptor occupation.

The role of caffeine-sensitive Ca2+ stores in agonist- and inositol 1,4,5-trisphosphate-induced Ca2+ release from bovine adrenal chromaffin cells

In single bovine adrenal chromaffin cells loaded with fura-2, histamine, angiotensin II (AII) and caffeine elicited large transient increases of intracellular free Ca2+ concentration [( Ca2+]i) in the absence of external Ca2+, with peak amplitudes averaging 726 +/- 138 (n = 14), 710 +/- 102 (n = 21) and 830 +/- 100 nM (n = 30) respectively. A substantial portion of the agonist-induced rise in [Ca2+]i depended on Ca2+ release from caffeine-sensitive stores, as pretreatment with caffeine diminished subsequent agonist responses by 90-95%. Conversely, pretreatment with histamine or AII decreased subsequent caffeine responses by 100% and 90% respectively. The effects of caffeine most likely resulted from activation of a Ca(2+)-induced Ca(2+)-release (CICR) process, whereas histamine and AII initially acted through generation of Ins(1,4,5)P3. The relationship of Ins(1,4,5)P3- and caffeine-sensitive Ca2+ pools was studied by using alpha-toxin-permeabilized chromaffin cells. Evidence was found for three non-mitochondrial, ATP-dependent, Ca2+ pools: one exclusively sensitive to Ins(1,4,5)P3 (pool 1), a second sensitive to both Ins(1,4,5)P3 and caffeine (pool 2), and a third exclusively sensitive to caffeine (pool 3). The existence of pools 1 and 3, and the ability of agonists such as histamine to discharge pool 3 completely, supports a two-pool model in which a caffeine-sensitive CICR mechanism plays a major role in the generation of agonist-induced Ca2+ spikes in bovine chromaffin cells.

Agonist-dependent patterns of cytosolic Ca2+ changes in single bovine adrenal chromaffin cells: relationship to catecholamine release.

The patterns of agonist-induced elevations of cytosolic free Ca2+ ([Ca2+]i) were characterized and compared by the use of single adrenal chromaffin cells. Initial histamine- or angiotensin II (AII)-induced elevations of [Ca2+]i were equal in magnitude (peaks 329 +/- 20 [SE] and 338 +/- 46 nM, respectively). These initial increases of [Ca2+]i were transient, insensitive to either Gd3+ or removing external Ca2+, and were primarily the result of Ca2+ release from intracellular stores. After the initial peak(s) of [Ca2+]i, a second phase of moderately elevated [Ca2+]i was observed, and this response was sensitive to either Gd3+ or removing external Ca2+, supporting a role for Ca2+ entry. In most cases, the second phase of elevated [Ca2+]i was sustained during histamine stimulation but transient during AII stimulation. Maintenance of the second phase was a property of the agonist rather than of the particular cell being stimulated. Thus, individual cells exposed sequentially to histamine and AII displayed distinct patterns of [Ca2+]i changes to each agonist, regardless of the order of addition. Histamine also stimulated twice as much [3H]catecholamine release as AII, and release was completely dependent on external Ca2+. Therefore, the ability of histamine and AII to sustain (or promote) Ca2+ entry appears to underlie their efficacy as secretagogues. These data provide evidence linking agonist-dependent patterns of [Ca2+]i changes in single cells with agonist-dependent functional responses.

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)

Localization and heterogeneity of agonist-induced changes in cytosolic calcium concentration in single bovine adrenal chromaffin cells from video imaging of fura-2.

Temporal and spatial changes in the concentration of cytosolic free calcium ([Ca2+]i) in response to a variety of secretagogues have been examined in adrenal chromaffin cells using digital video imaging of fura-2-loaded cells. Depolarization of the cells with high K+ or challenge with nicotine resulted in a rapid and transient elevation of [Ca2+]i beneath the plasma membrane consistent with Ca2+ entry through channels. This was followed by a late phase in which [Ca2+]i rose within the cell interior. Agonists that act through mobilization of inositol phosphates produced an elevation in [Ca2+]i that was most marked in an internal region of the cell presumed to be the site of IP3-sensitive stores. When the same cells were challenged with nicotine or high K+, to trigger Ca2+ entry through voltage-dependent channels, the rise in [Ca2+]i was most prominent in the same localized region of the cells. These results suggest that Ca2+ entry through voltage-dependent channels results in release of Ca2+ from internal stores and that the bulk of the measured rise in [Ca2+]i is not close to the exocytotic sites on the plasma membrane. Analysis of the time courses of changes in [Ca2+]i in response to bradykinin, angiotensin II and muscarinic agonists showed that these agonists produced highly heterogeneous responses in the cell population. This heterogeneity was most marked with muscarinic agonists which in some cells elicited oscillatory changes in [Ca2+]i. Such heterogeneous changes in [Ca2+]i were relatively ineffective in eliciting catecholamine secretion from chromaffin cells. A single large Ca2+ transient, with a component of the rise in [Ca2+]i occurring beneath the plasma membrane, may be the most potent signal for secretion.

Spontaneous and secretagogue-induced changes in cytosolic free Ca concentration measured by microfluorimetry with fura-2 on single bovine adrenal chromaffin cells.

The concentration of cytosolic Ca2+ ([Ca]in) was examined in single bovine adrenal chromaffin cells by monitoring fura-2 fluorescence with microspectrofluorimetry. To see the correlation between [Ca]in and secretion, we also measured the rates of catecholamine (CA) secretion and 45Ca efflux from populations of cells. [Ca]in was constant in the majority of single cells, but the small oscillatory changes in [Ca]in were observed in a population of cells. These spontaneous Ca oscillations, when observed, disappeared either after removal of extracellular Ca2+ or by addition of D-600 or Mn2+, but still persisted in the presence of tetrodotoxin (TTX) or after removal of extracellular Na+. In the silent cells the Ca fluctuations were often induced by Bay-K-8644. The characteristics of Bay-K-8644-induced Ca fluctuations were very similar to those of spontaneous ones. Low concentrations of nicotine (1 microM), acetylcholine (ACh; 1-2 microM), or KCl (12.5 mM) often induced oscillations riding on a steady rise in [Ca]in. These changes were rapidly suppressed by removal of either extracellular Ca2+ or Na+, or by addition of either D-600 (methoxyverapamil) or TTX. A low concentration of ACh (1 microM) or KCl (12.5 mM) also increased the rate of 45Ca efflux, but substantial secretion was not detected. On the other hand, the sustained rise in [Ca]in was evoked by 0.1 mM ACh, 20 microM nicotine, or 30 mM KCl, which was suppressed by removal of extracellular Ca2+, but was little affected by TTX. A sustained increase in 45Ca efflux upon exposure to ACh was observed, possibly reflecting the sustained rise in [Ca]in. ACh also stimulated CA secretion, which was faded out during the prolonged application. Veratridine, a Na channel activator, caused repetitive sequence of Ca transients followed by a sustained rise in [Ca]in. These results, together with the previous electrophysiological findings, suggest that: (1) the spontaneous Ca fluctuations are closely associated with occurrence of spontaneous Ca2+ and Na+ action potentials; (2) the rise in [Ca]in induced by a low concentration of nicotinic agonists of KCl is mediated by Na+ action potentials as well as gradual membrane depolarizations; (3) the oscillatory changes subsequent to a rise in [Ca]in reflect fluctuations in Ca2+ influx through the Ca2+ channels; (4) the critical [Ca]in needs to be attained before the CA secretion takes place.