Calcium-Dependent Binding of Cytosolic Proteins by Chromaffin Granules from Adrenal Medulla

Abstract: Purified chromaffin granules from bovine adrenal medulla bound a small group of medullary cell cytosol proteins at micromolar levels of Ca²⁺ and physiological levels of K⁺, Mg²⁺, and Mg-ATP. The bound proteins had molecular weights of 33,000-37,000 and 70,000-71,000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and did not correspond with any previously reported cytosolic components of chromaffin cells. The new proteins were eluted from intact granules or resealed granule membranes at 0.1 μ M Ca²⁺; binding was half-maximal at 2.6 μ M . Adsorption and elution in this manner resulted in a high degree of purification of the new proteins that were minor components of the original cytosol. Partially purified fractions enriched in the 33,000-37,000 and 70,000-71,000 proteins bound ⁴⁵Ca²⁺ at submicromolar levels in the presence of millimolar Mg²⁺. Calmodulin was also bound by the granule membranes and was present in trace amounts in cytosol eluates from granules, but it did not bind to the new proteins in the presence of calcium ions. The possible significance of the new proteins to calcium-mediated secretion from chromaffin cells is discussed.     

Characterization of Two Chromaffin Cell Populations Isolated from Bovine Adrenal Medulla

Cultures of chromaffin cells isolated from the bovine adrenal medulla have been extremely useful for investigating secretory mechanisms, but such cultures used up to the present time represent mixed populations of adrenergic and noradrenergic cells. This report describes how, with slight modifications to standard procedures, two separate chromaffin cell populations may be separated from bovine adrenal medullae. These two cell fractions have been characterized by biochemical, immunocytochemical, and morphological techniques as enriched populations of adrenergic or noradrenergic cells, respectively. The adrenergic cell-enriched fraction consists of greater than 90% adrenergic cells, whereas the noradrenergic cell-enriched fraction contains greater than 60% noradrenergic cells. We also demonstrate that these cells may be cultured with their secretory machinery intact: analysis of secreted catecholamines from nicotine- or high K+ concentration-stimulated cells cultured from each fraction confirms that adrenaline is the major catecholamine secreted by one fraction, whereas noradrenaline is mainly secreted by the other.     

Functional Aspects of Calcium Channels of Splanchnic Neurons and Chromaffin Cells of the Rat Adrenal Medulla

Effects of the inorganic calcium channel blockers zinc, manganese, cadmium, and nickel on secretion of catecholamines from the perfused adrenal gland of the rat were investigated. Secretion of catecholamines evoked by splanchnic nerve stimulation (1 and 10 Hz) was not affected by nickel (100 microM), partially blocked (50%) by cadmium (100 microM), and almost completely blocked (90%) by zinc (1 mM) or manganese (2 mM). A combination of nickel and cadmium inhibited nerve stimulation-evoked secretion by 80-90%. Catecholamine secretion evoked by direct stimulation of chromaffin cells by acetylcholine (50 micrograms), nicotine (5 microM), muscarine (50 micrograms), and K+ (17.5 mM) was not blocked by either cadmium, nickel, or their combination. However, zinc and manganese almost abolished nicotine- and K(+)-evoked secretion of catecholamines. None of the above agents had any effect on the secretion evoked by muscarine. Acetylcholine-evoked secretion of catecholamines was only partially reduced (50%) by zinc and manganese. We draw the following conclusions from the above findings: (a) cadmium plus nickel selectively blocks the calcium channels of splanchnic neurons but has no effect on calcium channels of the chromaffin cells; (b) zinc and manganese do not discriminate between calcium channels of neurons and calcium channels of chromaffin cells; (c) partial inhibition of acetylcholine-evoked secretion by inorganic calcium channel blockers is consistent with the idea that activation of nicotinic receptors increases Ca2+ influx, and activation of muscarinic receptors mobilizes intracellularly bound Ca2+, which is not affected by calcium channel blockers.     

Arachidonic Acid Liberated by Diacylglycerol Lipase Is Essential for the Release Mechanism in Chromaffin Cells from Bovine Adrenal Medulla

Chromaffin cells from bovine adrenal medulla secrete catecholamines on stimulation with acetylcholine. In addition to the activation of the phosphatidylinositol cycle, arachidonic acid is generated, which was thought to be the result of phospholipase A2 activation. We have demonstrated in isolated plasma membranes of these cells that arachidonic acid is generated by a two-step reaction of diacylglycerol and monoacylglycerol lipase splitting diacylglycerol, which originates from the action of phospholipase C on phosphatidylinositols. No phospholipase A2 activity could be detected in plasma membranes so far. External addition of arachidonic acid increases the release in the absence and in the presence of agonist. Inhibition of the diacylglycerol lipase by RHC 80267 suppresses the catecholamine release, which is restored on addition of arachidonic acid. This effect, however, is reversed by lipoxygenase inhibitors, indicating that it is not arachidonic acid itself, but one of its lipoxygenase products, that is essential for inducing exocytosis.     

Uptake of Nucleotides and Catecholamines by Chromaffin Granules from Pig and Horse Adrenal Medulla

Abstract: The uptake of nucleotides and Catecholamines into chromaffin granules from adrenals of pigs and horses is similar to that previously seen in bovine chromaffin granules. The rate of [³H]ATP uptake at 2 mM-ATP concentration was 0.42 ± 0.06 and 0.15 ± 0.02 nmol/mg protein/min for pig and horse granules, respectively. The apparent K_m's were 1.37 mM for pig granules, 0.89 mM for horse granules, and 1.2 mM for ox granules. The sensitivity of the uptake for nucleotides and catecholamine to specific inhibitors was found to be similar in granules from pig and ox, indicating that the same mechanisms of uptake are involved in both species.     

Secretion of Catecholamines from Individual Adrenal Medullary Chromaffin Cells

Abstract: Catecholamine secretion has been measured with electrochemical techniques from isolated, single adrenal medullary chromaffin cells with carbon-fiber microelectrodes. The electrode tip, which is of similar dimensions to the cell, is placed adjacent to the cell to enable the measurement of local secretion. Secretion is caused by exposing the cell to nanoliter volumes of solution containing nicotinic receptor agonists or depolarizing agents. The identification of secreted substances is made with cyclic voltammetry at both bare electrodes and electrodes coated with a perfluorinated cationexchange polymer. Catecholamine secretion is induced by nicotine (10–500 μ M ), carbamylcholine (1 m M ), and K⁺ (60 m M ). All agents that induce secretion lead to a broad envelope of secreted catecholamines on which sharp concentration spikes are superimposed. The concentration spikes can be monitored with a time resolution of tens of milliseconds when the electrodes are used in the amperometric mode. Release induced by nicotine and K⁺ is inhibited by Cd²⁺ (0.5 m M ), and hexamethonium selectively blocks the nicotineinduced secretion. The actions of nicotine are found to continue for a longer period of time than those of the other secretagogues tested.     

Cytokine Interactions with Adrenal Medullary Chromaffin Cells

It is generally accepted that a bi-directional or reciprocal interaction occurs between the immune and neuroendocrine systems, and that this relationship is important for the appropriate physiological functioning of both systems. Similarly, an imbalance in this relationship may contribute to a number of pathologies, most notably those relating to stress. The aim of this article is to consider the interaction of cytokines with the adrenal medulla, a potentially important player in this relationship. The chromaffin cells of the adrenal medulla release catecholamines and a range of biologically active peptides in response to a wide variety of stress-related signals. A growing body of evidence indicates that this stress response is influenced by, and in turn has influence upon, immune signalling. This brief review will focus primarily on the best-described adrenal medullary active cytokines, namely interferon-α, interleukin-6, interleukin-1α/β and tumour necrosis factor-α. In each case, three key issues will be addressed: the physiologically relevant source of the cytokine; the intracellular signalling events arising from activation of its receptor and finally the cellular consequences of such activation in terms of modulation of gene expression and the secretory output of the chromaffin cells.     

Purine Nucleotide Synthesis in Adrenal Chromaffin Cells

Abstract: The synthesis of purine nucleotides from the salvage precursors adenine and adenosine, and from the de novo precursors formate and glycine, was studied in isolated adrenal chromaffin cells. Both [8-¹⁴C]adenine and [8-¹⁴C]adenosine from extracellular medium are effectively incorporated into intracellular nucleotides. [¹⁴C]Formate and [U-¹⁴C]glycine are also incorporated, but de novo synthesis is clearly lower than synthesis from salvage precursors, although similar to de novo synthesis in liver. The enzymes responsible for adenine and adenosine salvage, adenine phosphoribosyltransferase and adenosine kinase, were purified about 1,500-fold. Both enzymes are mainly cytosolic and exhibit a similar molecular weight of around 42,000. The results suggest that chromaffin cells can replenish their intracellular nucleotides lost during the secretory event by an active synthesis from salvage and de novo precursors.     

Intracellular pH and Catecholamine Secretion from Bovine Adrenal Chromaffin Cells

To study the role of intracellular pH (pHi) in catecholamine secretion and the regulation of pHi in bovine chromaffin cells, the pH-sensitive fluorescent indicator [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] was used to monitor the on-line changes in pHi. The pHi of chromaffin cells at resting state is approximately 7.2. The pHi was manipulated first by incubation of the cells with NH4+, and then the solution was replaced with a NH4(+)-free solution to induce acidification of the cytoplasm. The pHi returned toward the basal pH value after acidification within 5-10 min in the presence of Na+ or Li+, but the pHi stayed acidic when Na(+)-free buffers were used or in the presence of amiloride and its analogues. These results suggest that the pH recovery process after an acid load is due to the Na+/H+ exchange activity in the plasma membrane of the chromaffin cells. The catecholamine secretion evoked by carbachol and Na+ removal was enhanced after the cytoplasm had been made more acidic. It appears that acidic pH favors the occurrence of exocytosis.     

Processing of Proenkephalin in Adrenal Chromaffin Cells

The processing of proenkephalin was studied using [35S]methionine pulse-chase techniques in primary cultures of bovine adrenal medullary chromaffin cells. Following radiolabeling, proenkephalin-derived peptides were extracted from the cells and separated by reverse-phase HPLC. Fractions containing proenkephalin fragments were digested with trypsin and carboxypeptidase B to liberate Met-enkephalin sequences and subjected to a second HPLC step to demonstrate association of radiolabel with Met-enkephalin. Processing of proenkephalin is complete within 2 h of synthesis, suggesting completion at or soon after incorporation into storage vesicles. Pretreatment of the cells with nicotine, histamine, or vasoactive intestinal peptide to enhance the rate of proenkephalin synthesis failed to alter the time course of processing and had minimal effects on the distribution of products formed. Addition of tetrabenazine, an inhibitor of catecholamine uptake into chromaffin vesicles, during radiolabeling and a 6-h chase period caused enhanced proenkephalin processing. These results suggest that the full range of proenkephalin fragments normally found in the adrenal medulla (up to 23.3 kDa) represents final processing products of the tissue and that termination of processing may depend on the co-storage of catecholamines.     

Characterization of Histamine-Induced Catecholamine Secretion from Bovine Adrenal Medullary Chromaffin Cells

Histamine activation of H1 receptors stimulates 3H release from cultured bovine adrenal chromaffin cells preloaded with [3H]noradrenaline. The initial (1-min) release induced by a high concentration of histamine was unaffected by the removal of extracellular Ca2+, whereas the more sustained response (10 min) was largely inhibited. In contrast, release induced by nicotine was dependent on extracellular Ca2+ at all times. The protein kinase inhibitor staurosporine inhibited both the initial and sustained (10-min) phases of histamine-induced release (IC50 in the region of 200 nM) but was ineffective against a direct depolarizing stimulus (56 mM K+). In contrast, the calmodulin antagonist trifluoperazine was equally effective against both stimuli. These data indicate that although a staurosporine-sensitive event (perhaps involving protein kinase C) is essential for coupling histamine receptor activation to the release processes, it is not essential for exocytosis itself. A further distinction between histamine- and depolarization-induced release was demonstrated by the differential effect of the guanine nucleotide-binding protein inhibitor pertussis toxin. Pretreatment with pertussis toxin (0.1 microgram/ml for 16 h) enhanced depolarization-induced release by approximately 1.5-fold. This pertussis toxin pretreatment was, however, approximately twofold as effective in potentiating histamine-evoked release. Thus, the characteristics of the histaminergic response are distinct from those of a depolarizing stimulus, perhaps indicating the involvement of different mechanisms in the release process.     

Serotonin Modulates Nicotinic Responses of Adrenal Chromaffin Cells

Abstract: 5-Hydroxytryptamine (5-HT) specifically and reversibly inhibits nicotine-induced currents and catecholamine release in bovine adrenal chromaffin cells in culture. Pharmacological analysis indicates that the inhibition is not mediated by known 5-HT receptor subtypes. The inhibition is noncompetitive over a range of nicotine concentrations between 1 and 100 μM. Preincubation with either 5-HT or substance P significantly protects the response from nicotine-induced desensitization. It is concluded that 5-HT inhibits nicotinic acetylcholine receptors on bovine adrenal chromaffin cells, probably by binding to a noncompetitive site on the receptor itself. Because both blood and the chromaffin cells contain 5-HT, the inhibition provides an opportunity for negative control of catecholamine secretion from the adrenals.     

Dynamin and Myosin Regulate Differential Exocytosis from Mouse Adrenal Chromaffin Cells

Neuroendocrine chromaffin cells of the adrenal medulla represent a primary output for the sympathetic nervous system. Chromaffin cells release catecholamine as well as vaso- and neuro-active peptide transmitters into the circulation through exocytic fusion of large dense-core secretory granules. Under basal sympathetic activity, chromaffin cells selectively release modest levels of catecholamines, helping to set the “rest and digest” status of energy storage. Under stress activation, elevated sympathetic firing leads to increased catecholamine as well as peptide transmitter release to set the “fight or flight” status of energy expenditure. While the mechanism for catecholamine release has been widely investigated, relatively little is known of how peptide transmitter release is regulated to occur selectively under elevated stimulation. Recent studies have shown selective catecholamine release under basal stimulation is accomplished through a transient, restricted exocytic fusion pore between granule and plasma membrane, releasing a soluble fraction of the small, diffusible molecules. Elevated cell firing leads to the active dilation of the fusion pore, leading to the release of both catecholamine and the less diffusible peptide transmitters. Here we propose a molecular mechanism regulating the activity-dependent dilation of the fusion pore. We review the immediate literature and provide new data to formulate a working mechanistic hypothesis whereby calcium-mediated dephosphorylation of dynamin I at Ser-774 leads to the recruitment of the molecular motor myosin II to actively dilate the fusion pore to facilitate release of peptide transmitters. Thus, activity-dependent dephosphorylation of dynamin is hypothesized to represent a key molecular step in the sympatho-adrenal stress response.     

Stability of Bovine Adrenal Medulla Cells in Culture

The functional stability of primary cultures of adrenal medulla cells was investigated. Isolated cells were prepared by treatment of bovine adrenal glands with collagenase followed by purification on Percoll density gradients and were maintained in Dulbecco's medium containing 10% fetal calf serum. Within 12 h after plating on plastic culture dishes, the cells became firmly attached and exhibited good survival for periods of time up to 3 weeks, as indicated by their morphology using light and electron microscopy, by maintenance of their content of catecholamines, tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine N-methyltransferase, and their ability to respond to secretagogues. During the first 10 days to 2 weeks in culture there was little or no change in any of these parameters. During the 3rd week there were progressive losses of catecholamine and enzyme activities and increased vacuolization of medullary cells. The cells synthesized protein and RNA with no apparent loss in activities over the period studied, but did not incorporate [3H]thymidine into PCA-precipitable material. The cells responded to secretagogues and secretory antagonists similarly to isolated perfused adrenal glands. The studies described here demonstrate that primary cultures of adrenal medulla cells provide an excellent experimental system for obtaining more detailed information on stimulus-secretion coupling and other functional aspects of the adrenal medulla.     

Caffeine-Sensitive Calcium Stores in Bovine Adrenal Chromaffin Cells

Caffeine was used to study the intracellular Ca2+ pools of bovine chromaffin cells. Its effects on cytosolic Ca2+ concentration ([Ca2+]i) were examined using fura-2. Caffeine caused a transient increase in [Ca2+]i in the presence or absence of extracellular Ca2+. In the former case, the caffeine-induced [Ca2+]i increase was higher and stayed above the basal value for several minutes. In the latter case, the [Ca2+]i rise was lower and fell to the basal level within 1 min. These results suggest that caffeine increases [Ca2+]i by causing both Ca2+ influx and Ca2+ release from intracellular pools. In the absence of extracellular Ca2+, ionomycin but not caffeine caused a further increase in [Ca2+]i in cells that had been treated with caffeine. Apparently there are at least two intracellular Ca2+ pools, only one of which is sensitive to caffeine. The caffeine-induced [Ca2+]i rise became smaller when the cells were pretreated with the inositol trisphosphate-generating agonists, methacholine and bradykinin. In addition, methacholine was unable to initiate a [Ca2+]i transient after the cells had been treated with caffeine. The results indicate that the caffeine-sensitive Ca2+ pools overlap with the inositol trisphosphate-sensitive pool and that the size of the latter pool is smaller than that of the former. The caffeine-sensitive Ca2+ pools were refilled after high K+ treatment, which suggests that the caffeine-sensitive Ca2+ pools may be important in buffering the cytosolic Ca2+. The effect of caffeine on [Ca2+]i is not due to inhibition of phosphodiesterase. Our results support a Ca2+ entry model in which depletion of intracellular Ca2+ pools controls the rate of Ca2+ entry across the plasma membrane.     

Paracrine Role of GABA in Adrenal Chromaffin Cells

The function of GABA in the adrenal medulla is still controversial. We will review experimental results in vivo and in vitro in adrenal chromaffin cells of various mammals to clarify what has been elucidated and what still remains to be settled.     

Regulation of Proenkephalin Synthesis in Adrenal Medullary Chromaffin Cells

The synthesis of proenkephalin was assessed in primary cultures of bovine adrenal medullary chromaffin cells by incubation of the cells with [35S]methionine, digestion of proenkephalin-derived peptides with trypsin and carboxy-peptidase B, and quantitation of radioactivity incorporated into Met-enkephalin following reversed-phase HPLC. Nicotine, histamine, and vasoactive intestinal peptide each enhanced the rate of proenkephalin synthesis approximately 10-fold when examined between 16 and 32 h after the drug or hormone addition. Inclusion of nifedipine (1 microM) partially blocked the stimulatory effect of nicotine, but not that of vasoactive intestinal peptide or histamine, or proenkephalin synthesis. Theophylline, tetrabenazine, and angiotensin II also increased the rate of proenkephalin synthesis (three- to eight-fold). These increases in the apparent rate of proenkephalin synthesis were not attributable to altered [35S]methionine specific radioactivity or rates of turnover and did not reflect similar increases in total protein synthesis. The half-life for turnover of Met-enkephalin sequences was 3-4 days in the cultured chromaffin cell. These studies directly show that proenkephalin synthesis is the primary regulatory step in control of chromaffin cell opioid peptide content.     

Multiple Forms of Endocytosis In Bovine Adrenal Chromaffin Cells

We studied endocytosis in chromaffin cells with both perforated patch and whole cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric catecholamine detection. We found that chromaffin cells exhibit two relatively rapid, kinetically distinct forms of stimulus-coupled endocytosis. A more prevalent “compensatory” retrieval occurs reproducibly after stimulation, recovering an approximately equivalent amount of membrane as added through the immediately preceding exocytosis. Membrane is retrieved through compensatory endocytosis at an initial rate of ~6 fF/s. Compensatory endocytotic activity vanishes within a few minutes in the whole cell configuration. A second form of triggered membrane retrieval, termed “excess” retrieval, occurs only above a certain stimulus threshold and proceeds at a faster initial rate of ~248 fF/s. It typically undershoots the capacitance value preceding the stimulus, and its magnitude has no clear relationship to the amount of membrane added through the immediately preceding exocytotic event. Excess endocytotic activity persists in the whole cell configuration. Thus, two kinetically distinct forms of endocytosis coexist in intact cells during perforated patch recording. Both are fast enough to retrieve membrane after exocytosis within a few seconds. We argue that the slower one, termed compensatory endocytosis, exhibits properties that make it the most likely mechanism for membrane recycling during normal secretory activity.     

Formaldehyde-Induced Fluorescence as a Means for Differentiating Epinephrine Cells from Norepinephrine Cells in Adrenal Medulla

The Falck and Hi Harp technique for the cellular localization of catecholamines by formaldehyde-induced fluorescence was applied to rat, mouse, and Syrian hamster adrenal. Some medullary cells revealed an unexpected orange-brown fluorescence. In guinea pig or rabbit adrenals, which store predominantly epinephrine, orange-brown fluorescence was not readily observed. It was found in Syrian hamsters only at the medullary periphery, where norepinephrine-producing cells are known to occur. Orange-brown fluorescence was depleted by administration of reserpine and intensified by nialamide plus DOPA. The same cell clusters which stained specifically for norepinephrine with ferric ferricyanide were found in adjacent sections to exhibit orange-brown fluorescence. Only by reducing the temperature of the formaldehyde reaction could sections of hamster adrenal showing only yellow-green fluorescence be obtained. These data suggest that the orange-brown fluorescence might result from' polymerization, oxidation, or both, of the isoquinoline produced by the norepinephrine-formaldehyde reaction under conditions slightly more vigorous than optimal and in the presence of high concentrations of norepinephrine     

Inhibition of Catecholamine Secretion from Adrenal Medulla Cells by Neurotoxins and Cholinergic Antagonists

Abstract: The effects of several neurotoxins and cholinergic antagonists on the nicotine-induced secretion of catecholamines by adrenal medulla cells in culture were investigated. Aconitine, veratridine, and batrachotoxin, in the presence of 1 μ m -tetrodotoxin inhibited the nicotine-stimulated secretion of catecholamines in a dose-dependent manner in Locke's solution. In Na⁺-free sucrose medium, tetrodotoxin was not required to inhibit the stimulatory effects of aconitine, veratridine, and batrachotoxin, and these agents by themselves inhibited the nicotine-stimulated secretion of catecholamines. Scorpion venom, which also increases the flux of Na⁺ through tetrodotoxin-sensitive channels, was not an effective inhibitor of nicotine-stimulated secretion. Histrionicotoxin, atropine, hexamethonium, and decamethoniun–as well as the Na⁺-channel activators–noncompetitively inhibit nicotine-stimulated secretion. The effects of these agents on nicotine-stimulated secretion appear similar to their effects on the inhibition of depolarization at the neuromuscular junction. Reversibility studies suggest that the stimulatory and inhibitory sites of the neurotoxins are different, while studies in Na⁺-free media suggest that tetrodotoxin-insensitive sodium channels are not involved in the inhibitory effect of the neurotoxins. A possible site of action for the inhibitory effects of the neurotoxins. A possible site of action for the inhibitory effects of the neurotoxins is the nicotinic-receptor-associated ion channel.