Evidence that the secreting adrenal chromaffin cell releases catecholamines directly from ATP-rich granules

1. Cats' adrenal glands were perfused with Locke's solution and stimulated through the splanchnic nerves or by acetylcholine.

2. In response to such stimulation there appeared in the venous effluent, in addition to catecholamines, large amounts of AMP and adenosine and smaller amounts of ATP and ADP. Like the catecholamines, these substances had their origin in the chromaffin cells as was shown by their failure to appear when the splanchnic nerves were stimulated during perfusion with drugs blocking the adrenal synapses.

3. During stimulation the ratio of catecholamines: ATP and metabolites in the venous effluent corresponded closely with the reported ratio of catecholamines: adenine nucleotides in the `heavy' chromaffin granules.

4. Adenine nucleotide appeared in the adrenal effluent pari passu with catecholamines within a second or two of beginning stimulation.

5. It is concluded that the nucleotide-rich granules are the immediate source of catecholamines released from the stimulated adrenal chromaffin cell, and that the other two intracellular `pools' that have been described, nucleotide-poor and `free' cytoplasmic catecholamines, contribute little or not at all.

     

Analysis of the role of cyclic adenosine 3′,5′-monophosphate in catecholamine release

1. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels and catecholamine release were measured in cat adrenal glands perfused in situ with Locke solution.2. Cyclic AMP was present in the medulla in an amount which represented approximately one fifth of that present in the cortex.3. Perfusion with acetylcholine (ACh) or nicotine increased cyclic AMP both in the intact adrenal and in the perfusate. The time course of the changes in tissue cyclic AMP during stimulation was out of phase with the time course of catecholamine release. Maximal increases in cyclic AMP were not manifest until after 8 min of exposure to the secretogogue, whereas maximal rates of secretion occurred during the first minute.4. Theophylline (0.5 mM) increased basal and stimulated adrenal cyclic AMP levels, but did not potentiate the secretory response to ACh or nicotine.5. Perfusion with cyclic AMP or its dibutyryl derivative (0.2-4 mM) failed to effect a consistent or significant increase in the rate of catecholamine release and was unable to potentiate the secretory response to a submaximal concentration of ACh or calcium.6. The results suggest that, unlike calcium, cyclic AMP is not a direct mediator of medullary secretion.     

Glucose does not affect catecholamine stimulus-secretion coupling in rat adrenal medulla: relationship to low changes in osmolarity and to insulin

Glucose levels were analyzed to see whether they directly affect the catecholamine release from chromaffin cells. We incubated isolated adrenal medullae of rats in Krebs-Hepes modified solutions with several glucose concentration, in the presence and absence of carbachol or insulin. Transfer of the medulla from a solution with 11.1 mM of glucose to a 0.56 mM one caused an increase in catecholamine secretion. Relative increase in change of glucose levels from 25 to 0.56 mM and from 50 to 0.56 mM enhanced the effect mentioned above. An inhibitory effect was detected after transfer of the medulla from 0.56 mM to 50 mM glucose. However, correction of solution osmolarity with mannitol or NaCl switched back catecholamine secretion to basal levels in all groups, and correction of solution osmolarity with sucrose indicated an impairment of catecholamine release. No difference was observed in stimulated catecholamine secretion (100 microM of carbachol) at all glucose levels. Further, the presence of insulin did not affect catecholamine secretion in all groups. Our results suggest that in isolated adrenal medullae of rats (1) glucose or variations in glucose levels do not affect catecholamine released; (2) isolated adrenal medulla of rat was highly sensitive to hyperosmolarity and extremely sensitive to hyposmolarity; (3) Insulin had no acute direct effect on catecholamine secretion in isolated adrenal medullae of rats.     

The effect of ouabain on the secretion of catecholamines and on the intracellular concentration of potassium

1. Perfusion of bovine adrenal glands with 0.1 mM ouabain is accompanied by a doubling of the spontaneous rate of release of catecholamines and by a 66% increase in the response to stimulation with carbamylcholine.2. Perfusion with ouabain (0.1 mM) reduces the intracellular content of potassium from 106 to 63 mM.3. The effect of ouabain on catecholamine secretion is dependent upon the presence of Ca(2+) ions in contrast to the effect upon the potassium content which is not dependent upon the presence of calcium.4. Ouabain has no effect on the release of catecholamines from isolated chromaffin granules both in the presence and absence of Ca(2+) ions.5. A possible relationship between the secretion of catecholamines and the concentration gradient of potassium across the cell membrane is discussed.     

The metabolic requirements from catecholamine release from the adrenal medulla

1. The metabolic requirements for catecholamine secretion elicited by acetylcholine or by calcium plus high K(+) were studied on acutely denervated perfused cat adrenal glands.2. Glucose-deprivation plus anoxia caused an increase in the spontaneous catecholamine output from adrenal glands perfused with normal Locke solution, which was abolished by the removal of calcium from the perfusion medium.3. Anoxia plus glucose-deprivation did not depress the secretory response to repeated exposures of a low concentration of acetylcholine, but did depress the response to a higher concentration of acetylcholine. Glucose-deprivation and nitrogen, when imposed either separately or together, did not inhibit total catecholamine output in response to calcium. Differential analysis of the calcium-evoked secretion showed that during anoxia, catecholamine output was maintained primarily by adrenaline secretion.4. Cyanide (0.2 mM) potentiated the secretory response to calcium in the presence of glucose, but when glucose was omitted from the perfusion medium, cyanide caused a gradual decline in calcium-evoked secretion. Iodoacetic acid (IAA) (0.2 mM) depressed the response to calcium by about 50% under aerobic conditions and by 90% under anaerobic conditions.5. The glycogen content of medullae was profoundly depleted under anoxic conditions.6. It is concluded that energy is required for the secretory action of calcium on medullary chromaffin cells. The energy may be derived from glycolysis or oxidative metabolism. A possible interaction between calcium and adenosine triphosphate acid (ATP) in eliciting catecholamine secretion is discussed.7. The alteration in the percent adrenaline and noradrenaline secreted during anoxia indicates that anoxia may regulate medullary catecholamine secretion through a peripheral, as well as a central mechanism.     

Voltage-dependent, pertussis toxin insensitive inhibition of calcium currents by histamine in bovine adrenal chromaffin cells

Histamine is a known secretagogue in adrenal chromaffin cells. Activation of G-protein linked H(1) receptors stimulates phospholipase C, which generates inositol trisphosphate leading to release of intracellular calcium stores and stimulation of calcium influx through store operated and other channels. This calcium leads to the release of catecholamines. In chromaffin cells, the main physiological trigger for catecholamine release is calcium influx through voltage-gated calcium channels (I(Ca)). Therefore, these channels are important targets for the regulation of secretion. In particular N- and P/Q-type I(Ca) are subject to inhibition by transmitter/hormone receptor activation of heterotrimeric G-proteins. However, the direct effect of histamine on I(Ca) in chromaffin cells is unknown. This paper reports that histamine inhibited I(Ca) in cultured bovine adrenal chromaffin cells and this response was blocked by the H(1) antagonist mepyramine. With high levels of calcium buffering in the patch pipette solution (10 mM EGTA), histamine slowed the activation kinetics and inhibited the amplitude of I(Ca). A conditioning prepulse to +100 mV reversed the kinetic slowing and partially relieved the inhibition. These features are characteristic of a membrane delimited, voltage-dependent pathway which is thought to involve direct binding of G-protein betagamma subunits to the Ca channels. However, unlike virtually every other example of this type of inhibition, the response to histamine was not blocked by pretreating the cells with pertussis toxin (PTX). The voltage-dependent, PTX insensitive inhibition produced by histamine was modest compared with the PTX sensitive inhibition produced by ATP (28% vs. 53%). When histamine and ATP were applied concomitantly there was no additivity of the inhibition beyond that produced by ATP alone (even though the agonists appear to activate distinct G-proteins) suggesting that the inhibition produced by ATP is maximal. When experiments were carried out under conditions of low levels of calcium buffering in the patch pipette solution (0.1 mM EGTA), histamine inhibited I(Ca) in some cells using an entirely voltage insensitive pathway. We demonstrate that activation of PTX insensitive G-proteins (most likely Gq) by H(1) receptors inhibits I(Ca). This may represent a mechanism by which histamine exerts inhibitory (in addition to previously identified stimulatory) effects on catecholamine release.     

Responses of the transmembrane potential coupled to the ATP-evoked catecholamine release in isolated chromaffin granules

Using suspensions of chromaffin granules isolated from bovine adreno-medullae, the effects of Mg2+.ATP on membrane potential and catecholamine release across membranes of granules were investigated. The release of catecholamine was monitored by the measurement of changes in endogenous fluorescence of the amines, while the fluorometric method with 3,3'-dipropylthiadicarbocyanine iodide (diS-C3-(5)) was applied to the measurement of membrane potential changes. The catecholamine release brought about by Mg2+.ATP was found to obey Michaelis-Menten kinetics with Km-value for Mg2+ of about 0.15 mM and to be inhibited by dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane-bound H+-ATPase. Concomitantly with such an amine release, depolarization of the granule membrane was observed to occur. The magnitude of this depolarization closely paralleled with the rate of the amine release and followed a saturation kinetics of Michaelis-Menten type with Km for Mg2+ of 0.21 mM and for ATP of 0.22 mM. Moreover, such a depolarization was competitively suppressed by DCCD. Based on these results, the role of the transmembrane potential change of granules in the catecholamine release was discussed.     

Ba2+-induced ATP release from adrenal medullary chromaffin cells is mediated by Ba2+ entry through both voltage- and receptor-gated Ca2+ channels

We have measured on-line the exocytotic secretion of ATP from adrenal medullary chromaffin cells induced by Ba2+ using a luciferin/luciferase assay. We have found that Ba2+-induced ATP release requires the entry of Ba2+ through either voltage- or receptor-gated Ca2+ channels. This conclusion is based on the observations that short preincubations with low concentrations of either nicotine or K+ greatly enhance Ba2+-induced ATP release and that this augmentation can be blocked with the nicotinic receptor antagonist, hexamethonium, and the Ca2+ antagonist, nifedipine, respectively. Moreover, both nicotine and K+ stimulate 133Ba2+ uptake, which in the case of K+ is inhibited by nifedipine. These results support the hypothesis that the cellular events leading to Ba2+-induced secretion coincide at least in part with the events leading to Ca2+-dependent exocytosis.     

Phosphorus-31 nuclear magnetic resonance studies of pig adrenal glands

We have used phosphorus-31 nuclear magnetic resonance to study the secretion of adenosine 5'-triphosphate (ATP) from the medulla of perfused pig adrenal glands. The resonances of the nucleotide pools in the chromaffin granules and cytoplasm are clearly resolved and therefore the intragranular and cytoplasmic processes involving ATP can be monitored simultaneously in the gland during secretion. Secretion of nucleotide during a 3 h continuous stimulation by infusion of acetylcholine chloride was monitored by the decrease in intensity of the intragranular ATP resonances. Up to 40% of the total intragranular nucleotide was released under these conditions. The rate of secretion decreased with duration of stimulation. No significant changes in the steady-state levels of cytoplasmic ATP or in oxygen consumption were observed. The intragranular pH in ischaemic glands was 5.52 + 0.15, while in glands which had been perfused until their cytoplasmic nucleotide levels had recovered, the intragranular pH was 5.76 +/- 0.16. These results provide the first estimate of the internal pH of the chromaffin granules in intact perfused adrenal glands and show that no net acidification occurs in the presence of cytoplasmic ATP. However, the isolated chromaffin granule possesses a proton-pumping adenosine 5'-triphosphatase which, in the presence of a permeant counter-ion, such as chloride, acidifies the granule interior. It is, therefore, suggested that in the intact cell, the cytoplasmic concentration of permeant counter-ions is too low to allow electrically neutral proton accumulation in the granules.     

Sodium ions inhibit the stimulant action of caffeine on catecholamine secretion from adrenal chromaffin cells of the guinea pig

Catecholamine secretion evoked by caffeine (40 mM) was markedly enhanced by replacing NaCl in the medium with sucrose or KCl in the absence, but not in the presence, of extracellular Ca2+ and Mg2+ in both perfused adrenal glands and isolated chromaffin cells of the guinea pig. The response to caffeine declined on repetition, but was restored completely after readmission of Ca2+. These results indicate that extracellular Na+ inhibits caffeine from stimulating catecholamine secretion, which may be mediated by a release of Ca2+ from intracellular storage sites in the adrenal chromaffin cells in the presence of extracellular Ca2+ and/or Mg2+.     

The distribution of calcium,magnesium, copper and iron in the bovine adrenal medulla

The subcellular distributions of calcium, magnesium, catecholamine and protein have been measured in four batches of bovine adrenal medullae; in addition, copper and iron have been measured in the chromaffin granules. Calcium is mainly found in chromaffin granules; magnesium, by contrast, is largely cytoplasmic. The stoichiometry of calcium, magnesium and catecholamines in the chromaffin granule matrix is given and their intragranular concentrations are calculated using measurements of the internal water space of the granules. The chromaffin granules are found to behave as osmometers when the sucrose concentration of the supporting medium is varied between 0.3 and 0.7m; about 30% of the intragranular water is osmotically inactive. Copper is a component of the enzyme dopamine β-hydroxylase; approximately 40% of granule copper is present within the chromaffin granule matrix, the rest being membrane-bound. Iron, a component of cytochrome b₅₆₁, is retained in the membrane; there is twice as much iron as copper in the granule membrane.It is not known how high concentrations of catecholamines and ATP are maintained inside chromaffin granules (or synaptic vesicles). It is possible that metal ions are required to stabilise complexes of high molecular weight. The chromaffin granules are a major reservoir of calcium in adrenal medulla cells.     

Increased secretory capacity of mouse adrenal chromaffin cells by chronic intermittent hypoxia: involvement of protein kinase C

Previous studies have shown that catecholamine secretion from the adrenal medulla plays a critical role in chronic intermittent hypoxia (CIH)-induced alterations in cardiovascular function. In the present study we examined the cellular mechanisms associated with the effects of CIH on adrenal chromaffin cell catecholamine secretion. Experiments were performed on adult male mice (C57/BL6) that were exposed to 1–4 days of CIH or to normoxia. Perforated patch electrical capacitance recordings were performed on freshly prepared adrenal medullary slices that permit separating the chromaffin cell secretion from sympathetic input. CIH resulted in a significant increase in the readily releasable pool (RRP) of secretory granules, and decreased stimulus-evoked Ca²⁺ influx. Continuous hypoxia (CH) either for 2.5 h (equivalent to hypoxic duration accumulated over 4 days of CIH) or for 4 days were ineffective in evoking changes in the RRP and Ca²⁺ influx. CIH activated PKC in adrenal medullae as evidenced by increased phosphorylation of PKC at Thr⁵¹⁴ and PKC inhibitors prevented CIH-induced increases in the RRP and restored stimulus-evoked attenuation of Ca²⁺ influx. CIH resulted in elevated thio-barbituric acid reactive substances (TBARSs, an index of oxidized proteins) and an antioxidant prevented CIH-induced changes in the RRP, suggesting the involvement of reactive oxygen species (ROS). These results demonstrate that CIH increases the RRP in adrenal chromaffin cells via ROS-mediated activation of PKC and suggest that CIH can directly affect the secretory capacity of chromaffin cells and contribute, in part, to elevated catecholamine levels.     

ATP-sensitive K+ transport in adrenal chromaffin granules

In the present study the influx of 86Rb+, a K+ analogue, was studied in mitochondria, microsomes and chromaffin granules prepared from adrenal gland medulla. The most active electrogenic 86Rb+ transport was found in the membrane fraction identified as chromaffin granules by marker enzyme estimation. The transport was found to be sensitive to ATP, ATP gamma S, ADP and to the triazine dyes, but not to AMP and cAMP. The inhibition induced by ATP was observed in the absence of externally added Mg2+, suggesting that a free nucleotide, rather than the ATP-Mg complex, was required for inhibition. Furthermore, the 86Rb+ influx was found to be inhibited by Mg2+ alone, but not by Ca2+ and antidiabetic sulfonylureas. The 86Rb+ influx was not stimulated by potassium channel openers. In conclusion, our results indicate that an electrogenic, ATP-sensitive potassium transport system operates in the chromaffin granule membrane.     

Prompt recovery of damaged adrenal medullae induced by salinomycin.

The morphologic changes in the adrenal medullae of rats treated with an ionophore antibiotic, salinomycin, are described. Male rats of approximately 7 wk of age were treated orally with a single dose of salinomycin at 80 mg/kg body weight. Following this treatment, the adrenal glands were examined, using immunohistochemistry, for neurofilament, laminin, fibronectin, and S-100 protein; the glands were also examined using transmission electron microscopy. One hour after the treatment, a karyopyknosis was observed in the clusters of affected chromaffin cells in which the neurofilament, laminin, and fibronectin were present. The lesions became progressively conspicuous between hours 5 and 10. Ultimately, the outcome was cell lysis. Five hours after salinomycin treatment, unaffected chromaffin cells strongly stained to tyrosine hydroxylase. At 10 hr, new chromaffin cells, which were irregular in shape with electron-dense cytoplasm (dark cell), that were strongly stained for tyrosine hydroxylase appeared at the basement membrane site of the necrotic clusters, and these cells contained very few immature catecholamine granules of less than 80 nm. At 17 hr, the catecholamine granules increased in number and size to about 200 nm. The newly formed chromaffin cells grew within the clusters to fill in the medulla by 24 hr, and cytoplasmic granules progressively increased in number and size. The interstitial tissue was seen to be edematous at 5 hr. New capillaries were found in the adrenal medullae of both control and salinomycin-treated rats. The protruding chromaffin cells (protruding cells), which we previously described in normal rats, were also observed in salinomycin-treated rats, which suggests that holocrine secretion is performed in the adrenal medullae. The results indicated that the rat adrenal medullae have the ability to make a rapid recovery after an insult by salinomycin.     

Chromaffin granules in the rat adrenal medulla release their secretory content in a particulate fashion

Exocytosis is considered the main route of granule discharge in chromaffin cells. We recently provided ultrastructural evidence suggesting that piecemeal degranulation (PMD) occurs in mouse adrenal chromaffin cells. In the present study, we processed rat adrenal glands for transmission electron microscopy (TEM), and examined chromaffin cells for changes characteristic of PMD. Both adrenaline (A)- and noradrenaline (NA)-storing cells express ultrastructural features suggestive of a slow and particulate mode of granule discharge. In adrenaline-containing cells, some granules present enlarged dimensions accompanied by eroded or dissolved matrices. Likewise, a number of granules in NA-releasing cells show content reduction with variably expanded granule chambers. Dilated, empty granule containers are recognizable in the cytoplasm of both cell types. Characteristically, altered granules and empty containers are seen intermingled with normal, resting granules. In addition, chromaffin granules often show irregular profiles, with budding or tail-like projections of their limiting membranes. Thirty 150-nm-diameter membrane-bound vesicles with a moderately electron-dense or -lucent internal structure are observable in the cytoplasm of both cell types. These vesicles are seen among the granules and some of them are fused with the perigranule membranes in the process of attachment to or budding from the granules. These data add further support to the concept that PMD may be an alternative secretory pathway in adrenal chromaffin cells.     

Synaptic activation of rat adrenal medulla examined with a large photodiode array in combination with a voltage-sensitive dye.

The adrenal medulla is innervated by sympathetic preganglionic nerve fibers in the splanchnic nerve. Synaptic activation of the adrenal medulla causes catecholamine secretion which is known to be modified by various neuropeptides and other factors. To understand the neuronal control mechanism of catecholamine secretion, it is necessary to know the transfer function at the synapse and how it is affected by such factors. By using a large photodiode array in combination with a voltage-sensitive dye, membrane potential changes in a slice of the rat adrenal gland were recorded upon brief local electrical stimulation. Electrical signals were recorded only on the portion of the diode array corresponding to the medulla. In a typical record, a spike and an underlying slow potential were observed following a small deflection due to a presynaptic nerve action potential. Both the spike and slow potential were blocked in Ca(2+)-free solution or by hexamethonium, a nicotinic antagonist, but were not affected by atropine, a muscarinic antagonist. The slow potential was interpreted as a nicotinic synaptic potential in the chromaffin cells and the spike as a population action potential. A double pulse experiment revealed that the chromaffin cell action potential began to fail only when the stimulus interval was less than 50 ms (20 Hz). When the stimulus intensity was reduced, the minimal response was found to behave in an all-or-none fashion. This suggested that one nerve fiber is innervating a cluster of chromaffin cells, which may correspond to a previously histologically identified "complex" of cells [Hillarp (1946) Acta. anat. 4, Suppl. 1]. Each complex was innervated by approximately four nerve fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake of adenosine triphosphate by isolated adrenal chromaffin granules: A carrier-mediated transport

Partly purified chromaffin granules from bovine adrenal medulla were incubated in vitro with [³H]adenosine triphosphate (ATP). After several washes the particles were subjected to density gradient centrifugation. It was demonstrated that chromaffin granules had taken up radioactive material. Thin-layer chromatography revealed that more than 60% of the radioactivity was confined to [³H]ATP. Incubation experiments with a mixture of [³H]ATP and [³²P]ATP indicated that ATP was taken up by chromaffin granules as an intact molecule. The uptake of [³H]ATP was temperature dependent and linear up to 10 min. The rate of uptake depended upon the ATP concentration and levelled off at ATP concentrations above 2 mM. At this ATP concentration the rate was 0.268 nmol ATP/mg protein/min. The uptake of ATP was compared with that of catecholamines. Both processes were activated by Mg²⁺, but inhibited by N-ethylmaleimide and an uncoupler of oxidative phosphorylation. Atractyloside inhibited only the ATP-uptake, whereas reserpine only interfered with that of the catecholamines.It is suggested that this uptake mechanism for ATP is carrier-mediated and that it enables newly formed chromaffin granules to accumulate and to maintain a high nucleotide concentration, which is required for the formation of a storage complex with catecholamines.     

Autocrine/paracrine modulation of calcium channels in bovine chromaffin cells

 Applying 10-s pulses of 10 mM Ba²⁺ to resting or K⁺-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca²⁺ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr²⁺ or Ca²⁺ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca²⁺, Sr²⁺ and Ba²⁺. Ca²⁺ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I _Ca by 19%, I _Sr by 31%, and I _Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing prepulses (facilitation). Under continuous-flow conditions, 10 μM exogenous ATP reduced (about 50%) I _Ca, I _Sr and I _Ba similarly. On the other hand, the use of Na⁺ as a charge carrier through Ca²⁺ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating secretion from unpatched cells, by either 10 mM Ba²⁺, 100 μM acetylcholine or 70 mM K⁺, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca²⁺ channels through autocrine/paracrine mechanisms. Received: 29 June 1998 / Received after revision: 20 August 1998 / Accepted: 1 September 1998     

Role of ATP decrease in secretion induced by mitochondrial dysfunction in guinea-pig adrenal chromaffin cells

The mechanism related to mitochondrial dysfunction-induced catecholamine (CA) secretion in dispersed guinea-pig adrenal chromaffin cells was investigated using amperometry and confocal laser microscopy. Application of CCCP, which does not stimulate generation of reactive oxygen species (ROS), reversibly induced CA secretion, whereas application of either cyanide or oligomycin (OL), a stimulator for ROS, enhanced CA secretion to a smaller extent. The CCCP-induced secretion was abolished by removal of external Ca²⁺ ions and was markedly diminished by D600. The mitochondrial membrane potential, measured using rhodamine 123, was rapidly lost in response to CCCP, but did not change noticeably during a 3 min exposure to OL. Prior exposure to OL markedly facilitated depolarization of the mitochondrial membrane potential in response to cyanide. The mitochondrial inhibitors rapidly produced an increase in Magnesium Green (MgG) fluorescence in the absence of external Ca²⁺ and Mg²⁺ ions, an increase that was larger in the cytoplasm than in the nucleus. The rank order of potency in increasing MgG fluorescence among the inhibitors was similar to that in increasing secretion. Thus, mitochondrial inhibition rapidly decreases [ATP] and the mitochondrial dysfunction-induced secretion is not due to ROS generation or to mitochondrial depolarization, but is possibly mediated by a decrease in ATP.     

The role of energy metabolism in calcium-evoked secretion from the adrenal medulla

1. Experiments were carried out on cat adrenal glands perfused with Locke solution to study the effects of inhibition of metabolism on calcium-evoked catecholamine release.2. In the presence of sodium cyanide (CN, 0.2 mM), a low concentration of glucose (1 mM) prevented the gradual decline in the secretory response to sequential exposures to calcium. Furthermore, when the secretory response was almost completely blocked by perfusing with a glucose-deprived solution containing CN, restoration of secretion was correlated with the glucose concentration in the perfusion medium.3. In the presence of CN, 2-deoxyglucose blocked both the protective effect and the restorative effect of glucose. The deoxyglucose inhibition of the glucose-dependent restoration of secretion was antagonized by a higher concentration of glucose.4. Restoration of calcium-evoked secretion was also observed after the washout of CN. The extent of this restoration was not at all related to the glucose concentration and was not affected by various inhibitors of carbohydrate metabolism, including deoxyglucose.5. Analysis of adrenal glands which had been perfused first with a glucose-free solution containing CN and subsequently with the normal medium indicated that no discernible synthesis of catecholamines had taken place during the experimental procedures.6. The data provide further evidence that the action of calcium to trigger medullary secretion requires the presence of metabolic energy and support the hypothesis that an interaction between calcium and high-energy nucleotides is a step in the sequence of events leading to the extrusion of catecholamines from the chromaffin cell.