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Three divalent cations can elicit secretory responses in most neuroendocrine cells, including chromaffin cells. The extent to which secretion is elicited by the cations in intact depolarized cells was Ba 2+  > Sr 2+  ≥ Ca 2+ , contrasting with that elicited by these cations in permeabilized cells (Ca 2+  > Sr 2+  > Ba 2+ ). Current-clamp recordings show that extracellular Sr 2+ and Ba 2+ cause membrane depolarization and action potentials, which are not blocked by Cd 2+ but that can be mimicked by tetra-ethyl-ammonium. When applied intracellularly, only Ba 2+ provokes action potentials. Voltage-clamp monitoring of Ca 2+ -activated K + channels (K Ca ) shows that Ba 2+ reduces outward currents, which were enhanced by Sr 2+ . Extracellular Ba 2+ increases cytosolic Ca 2+ concentrations in Fura-2-loaded intact cells, and it induces long-lasting catecholamine release. Conversely, amperometric recordings of permeabilized cells show that Ca 2+ promotes the longest lasting secretion, as Ba 2+ only provokes secretion while it is present and Sr 2+ induces intermediate-lasting secretion. Intracellular Ba 2+ dialysis provokes exocytosis at concentrations 100-fold higher than those of Ca 2+ , whereas Sr 2+ exhibits an intermediate sensitivity. These results are compatible with the following sequence of events: Ba 2+ blocks K Ca channels from both the outside and inside of the cell, causing membrane depolarization that, in turn, opens voltage-sensitive Ca 2+ channels and favors the entry of Ca 2+ and Ba 2+ . Although Ca 2+ is less permeable through its own channels, it is more efficient in triggering exocytosis. Strontium possesses both an intermediate permeability and an intermediate ability to induce secretion.

Secretory vesicles of chromaffin cells are acidic organelles that maintain an increasing pH gradient towards the cytosol (5.5 vs. 7.3) that is mediated by V-ATPase activity. This gradient is primarily responsible for the accumulation of large concentrations of amines and Ca(2+), although the mechanisms mediating Ca(2+) uptake and release from granules, and the physiological relevance of these processes, remain unclear. The presence of a vesicular matrix appears to create a bi-compartmentalised medium in which the major fractions of solutes, including catecholamines, nucleotides and Ca(2+), are strongly associated with vesicle proteins, particularly chromogranins. This association appears to be favoured at acidic pH values. It has been demonstrated that disrupting the pH gradient of secretory vesicles reduces their rate of exocytosis and promotes the leakage of vesicular amines and Ca(2+), dramatically increasing the movement of secretory vesicles and triggering exocytosis. In this short review, we will discuss the data available that highlights the importance of pH in regulating the association between chromogranins, vesicular amines and Ca(2+). We will also address the potential role of vesicular Ca(2+) in two major processes in secretory cells, vesicle movement and exocytosis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na+/K+-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K+ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca2+ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) triggered by K+ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K+ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K+-elicited [Ca2+]c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca2+ load and facilitate the Ca2+-induced-Ca2+ release (CICR) component of the [Ca2+]c signal generated during K+ depolarisation. This could explain the potentiating effects of ouabain on exocytosis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Lead exposure is an important issue in the research of several toxic effects resulting from the biochemical interaction between this metal and the organism. Calcium is a fundamental mineral for the maintenance of the organism homeostasis where there is interaction between lead and calcium in metabolic pathways. Environmental lead exposure by verifying the usefulness and applicability of urinary calcium/creatinine ratio (UCa/Cr) in this context was evaluated. This was an extensive socio-demographic study of the nutritional profile, lead exposure biomarkers in blood and the urine and UCa/Cr ratio. The children studied were from a low socio-economic group characterized by unsatisfactory nutritional diet. Lead environmental exposure was shown by biomarkers, with UCa/Cr ratio having positive and significant correlations with both lead and delta-aminolevulinic acid in urine (ALA-U), without colinearity diagnosis. There was a strong association between calcium excretion and lead exposure as a result of linear regression construction models. In children, lead increases calcium excretion which is an additional risk to infantile health. Urinary calcium/creatinine ratio may be a useful tool in the biological monitoring of lead exposure and health promotion programs.

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient toward the cytosol (pH 5.5 versus 7.2) promoted by the V-ATPase activity. This gradient of pH is also responsible for the accumulation of amines and Ca2+ because their transporters use H+ as the counter ion. We have recently shown that alkalinization of secretory vesicles slowed down exocytosis, whereas acidification caused the opposite effect. In this paper, we measure the alkalinization of vesicular pH, caused by the V-ATPase inhibitor bafilomycin A1, by total internal reflection fluorescence microscopy in cells overexpressing the enhanced green fluorescent protein-labeled synaptobrevin (VAMP2-EGFP) protein. The disruption of the vesicular gradient of pH caused the leak of Ca2+, measured with fura-2. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that bafilomycin directly released Ca2+ from freshly isolated vesicles. The Ca2+ released from vesicles to the cytosol dramatically increased the granule motion of chromaffin- or PC12-derived granules and triggered exocytosis (measured by amperometry). We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of cytosolic Ca2+ and in two of the major functions of secretory cells, vesicle motion and exocytosis.

The relationship between cytosolic Ca2+ and catecholamine secretion during stimulus-secretion coupling has been examined at individual chromaffin cells isolated from the cow and rat. Vesicular catecholamine exocytosis was determined via amperometric measurements with carbon fibre microelectrodes and fura-2 was used for simultaneous fluorescent monitoring of cytosolic Ca2+ at the same cell. Individual secretory vesicles in cells from the two species were found to release similar amounts of catecholamine. In addition, the time courses for secretion from individual vesicles was similar with rat and bovine chromaffin cells. The total quantity of catecholamine released and the change in cytosolic Ca2+ were also similar in response to exposure to K+ (60 mM), 1,1-dimethyl-4-phenylpiperazinium (50 microM), and histamine (50 microM), although both responses were more prolonged following 1,1-dimethyl-4-phenylpiperazinium and histamine at bovine cells. Agents that mobilize intracellular Ca(2+)-stores such as methacholine, caffeine and bradykinin resulted in different cytosolic Ca2+ and exocytosis responses at the rat and bovine chromaffin cells. Results indicate a heightened Ca(2+)-store activity or a more filled state in chromaffin cells from the rat. The results of this study clearly show that single-cell techniques can be used to characterize stimulus-secretion coupling. The requirement for lower numbers of cells with these techniques means that chromaffin cells from rodents can be routinely employed. This can be advantageous to minimize biological variability which occurs with organs obtained from slaughter houses and enables the investigation of genetically-altered animals.

Histamine activation of H1 receptors elicited the release of adrenaline from in vitro perfused rat adrenal with an EC50 of 3 microM. Neither Na+ deprivation nor complete membrane depolarization abolished the histamine-mediated secretory response but it was partially dependent on extracellular Ca2+. Nitrendipine and BAY-K-8644 affected the release induced by histamine concentrations at over 3 microM. Delayed application of histamine pulses, after external Ca2+ removal, led to a decline in to a plateau at 50% of the initial release. Pretreatment with ionomycin abolished this Ca2+ deprivation-resistant component. These data suggest that secretion evoked by low concentrations of histamine occurs by mobilization of Ca2+ from internal stores whereas higher concentrations use Ca2+ from both intracellular and extracellular sources.

Dihydropyridines (+)-PN200-110 and (+/-)-Bay-K-8644 inhibit or potentiate, respectively, catecholamine release evoked by DMPP- or K-stimulation of perfused cat adrenal glands. Since both, secretion of adrenaline and noradrenaline are equally affected, and these two drugs specifically act on voltage-dependent chromaffin Ca channels, it seems that secretion of each amine from their respective cell is regulated by the same type of channel.

The rate of catecholamine release from cat adrenal glands perfused with Krebs solution containing 59 mM K declined exponentially during the first few minutes of depolarization. The rate of decline was considerably slower when Ca was substituted by Sr. The late addition of Ca, Sr or the Ca-channel activator BAY-K-8644 evoked a revival of secretion when catecholamine release was inactivated by prior K depolarization; the revival of secretion was independent of the depolarization time. These data demonstrate that inactivation of catecholamine release is specifically dependent on Ca; the modulatory role of Ca on secretion seems to be exerted at a step distal to the transmembraneous Ca channel.