Ricardo Borges

The Crucial Role of Chromogranins in Storage and Exocytosis Revealed Using Chromaffin Cells from Chromogranin A Null Mouse

Chromogranins (Cgs) are the major soluble proteins of dense-core secretory vesicles. Chromaffin cells from Chga null mice [chromogranin A knock-out (CgA-KO)] exhibited ∼30% reduction in the content and in the release of catecholamines compared with wild type. This was because of a lower secretion per single exocytotic event, rather than to a lower frequency of exocytotic events. Cell incubation with l-DOPA produced an increase in the vesicular amine content of wild-type, but not CgA-KO vesicles. In contrast, intracellular electrochemistry showed that l-DOPA produced a significantly larger increase in cytosolic amines in CgA-KO cells than in the wild type. These data indicate that the mechanisms for vesicular accumulation in CgA-KO cells were fully saturated. Patch-amperometry recordings showed a delayed initiation of the amperometric signal after vesicle fusion, whereas no changes were observed in vesicle size or fusion pore kinetics despite the smaller amine content. We conclude that intravesicular proteins are highly efficient systems directly implicated in transmitter accumulation and in the control of neurosecretion.

Chronic hypoxia up‐regulates α1H T‐type channels and low‐threshold catecholamine secretion in rat chromaffin cells

α1H T‐type channels recruited by β1‐adrenergic stimulation in rat chromaffin cells (RCCs) are coupled to fast exocytosis with the same Ca2+ dependence of high‐threshold Ca2+ channels. Here we show that RCCs exposed to chronic hypoxia (CH) for 12–18 h in 3% O2 express comparable densities of functional T‐type channels that depolarize the resting cells and contribute to low‐voltage exocytosis. Following chronic hypoxia, most RCCs exhibited T‐type Ca2+ channels already available at −50 mV with the same gating, pharmacological and molecular features as the α1H isoform. Chronic hypoxia had no effects on cell size and high‐threshold Ca2+ current density and was mimicked by overnight incubation with the iron‐chelating agent desferrioxamine (DFX), suggesting the involvement of hypoxia‐inducible factors (HIFs). T‐type channel recruitment occurred independently of PKA activation and the presence of extracellular Ca2+. Hypoxia‐recruited T‐type channels were partially open at rest (T‐type ‘window‐current’) and contributed to raising the resting potential to more positive values. Their block by 50 μm Ni2+ caused a 5–8 mV hyperpolarization. The secretory response associated with T‐type channels could be detected following mild cell depolarizations, either by capacitance increases induced by step depolarizations or by amperometric current spikes induced by increased [KCl]. In the latter case, exocytotic bursts could be evoked even with 2–4 mm KCl and spike frequency was drastically reduced by 50 μm Ni2+. Chronic hypoxia did not alter the shape of spikes, suggesting that hypoxia‐recruited T‐type channels increase the number of secreted vesicles at low voltages, without altering the mechanism of catecholamine release and the quantal content of released molecules.

Nitric Oxide Modulates a Late Step of Exocytosis

The effects of nitric oxide (NO) on the late phase of exocytosis have been studied, by amperometry, on Ba2+-stimulated chromaffin cells. Acute incubation with NO or NO donors (sodium nitroprusside, spermine-NO,S-nitrosoglutathione) produced a drastic slowdown of the granule emptying. Conversely, cell treatment with Nω-nitro-l-arginine methyl ester (a NO synthase inhibitor) or with NO scavengers (methylene blue, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium) accelerated the extrusion of catecholamines from chromaffin granules, suggesting the presence of a NO modulatory tone. The incubation with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine or zaprinast) or with the cell-permeant cGMP analog 8-bromo-cGMP, mimicked the effects of NO, suggesting the involvement of the guanylate cyclase cascade. NO effects were not related to changes in intracellular Ba2+. NO did not modify the duration of feet. Effects were evident even on pre-fusioned granules, observed under hypertonic conditions, suggesting that the fusion pore is not the target for NO, which probably acts by modifying the affinity of catecholamines for the intragranular matrix. NO could modify the synaptic transmitter efficacy through a novel mechanism, which involves the regulation of the emptying of secretory vesicles.

Multiple calcium channel subtypes in isolated rat chromaffin cells

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca2+ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of −80 mV, using 10 mM Ba2+ as the charge carrier, only high-voltage-activated (HVA) Ca2+ channels were found. Ba2+ currents (IBa) snowed variable sensitivity to dihydropyridine (DHP) Ca2+ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 μM and 10 μM respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca2+ channel agonist Bay K 8644 (1 μM) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of IBa was accompanied by a 20-mV shift in the activation curves for Ca2+ channels towards more hyperpolarizing potentials. Application of the conus toxin ω-conotoxin GVIA (GVIA; 1 μM) blocked 31% of IBa; blockade was irreversible upon removal of the toxin from the extracellular medium, ω-Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of IBa. ω-Conotoxin MVIIC (MVIIC; 5 μM) produced a 36% blockade of IBa; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca2+ channels. The sequential addition of supramaximal concentrations of furnidipine (10 μM), GVIA (1 μM), IVA (100 nM) and MVIIC (3 μM) produced partial inhibition of IBa, which were additive. Our data suggest that the whole cell IBa in rat chromaffin cells exhibits at least four components. About 50% of IBa is carried by L-type Ca2+ channels, 30% by N-type Ca2+channels and 15% by P-type Ca2+ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca2+channels account for 45% of the current, N-type carry 35% and L-type Ca2+ channels are responsible for only 20–25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species.

Effects of External Osmotic Pressure on Vesicular Secretion from Bovine Adrenal Medullary Cells

Secretion of catecholamines from individual vesicles of bovine adrenal medullary cells was studied with amperometry in media of various osmolarities and compared with results obtained in isotonic physiological buffer (315 mosM). Hypotonic solutions caused an increase in the number of amperometric spikes evoked by brief exposure to 5 mM Ba2+. Under moderate hypertonic conditions (630 mosM), individual vesicular events were decreased in frequency, and lower amounts were secreted per event. Furthermore, the events were temporally broadened relative to those observed during release in isotonic conditions. At 970 mosM, exposure to 5 mM Ba2+ evoked even smaller secretory events that resemble the prespike feature that has been attributed to the initial opening of the fusion pore. The lack of large spikes is not due to failure of Ba2+ entry because fura-2 fluorescence reveals an increase in intracellular divalent ions. After exposure to Ba2+ in hypertonic solution, spikes could be induced with isotonic solution transiently directed onto the cell, but this process was not accompanied by a change in the concentration of intracellular divalent ions. Thus, this procedure provides an unique opportunity to temporally separate exocytotic secretion from entry of divalent ions.

Automatic analysis for amperometrical recordings of exocytosis

Amperometry is a widely used technique for monitoring the secretion of catecholamines (CA) by exocytosis. The use of carbon fibre microelectrodes allows the on-line recording of CA released from a single secretory vesicle. Amperometric signals are generated by oxidation of the quantally released CA close to the electrode tip. Each event of exocytosis is called a secretory spike. Here we describe a program written for IGOR (Wavemetrics, Lake Oswego, OR, USA), which may be used to analyze amperometric signals off-line. The procedures allow, (i) digital filtering and analysis of the current noise, spike identification and calculation of spike kinetic parameters; (ii) spike review; (iii) pooling spikes and data to create galleries, tables and histograms of measured parameters which can be exported to a graphic format or files for further analysis.

Localized L-type calcium channels control exocytosis in cat chromaffin cells

Depolarizing 1-s pulses to 0 mV from a holding potential of −70 mV, induced whole-cell currents through Ca2+ channels (ICa) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 μM) reduced the peak current by 47% and the late current by 80%. ω-Conotoxin GVIA (CgTx, 1 μM) reduced the peak ICa by 42% and the late ICa by 55%. Pulses (10 s duration) with 70 mM K+/2.5 mM Ca2+ solution (70 K+/2.5 Ca2+), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca2+ concentration ([Ca2+]i from 0.1 to 2.21 μM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K+/2.5 Ca2+ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca2+]o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca2+ channels in cat chromaffin cells. It seems, howevever, that though extracellular Ca2+ entry through both channel types leads to similar increments of averaged [Ca2+]i, the control of catecholamine release is dominated only by Ca2+ entering through L-type Ca2+ channels. This supports the idea of a preferential segregation of L-type Ca2+ channels to localized “hot spots” in the plasmalemma of chromaffin cells where exocytosis occurs.

Intragranular pH rapidly modulates exocytosis in adrenal chromaffin cells.

Several drugs produce rapid changes in the kinetics of exocytosis of catecholamines, as measured at the single event level with amperometry. This study is intended to unveil whether the mechanism(s) responsible for these effects involve changes in the intravesicular pH. Cell incubation with bafilomycin A1, a blocker of the vesicular proton pump, caused both a deceleration in the kinetics of exocytosis and a reduction in the catecholamine content of vesicle. These effects were also observed upon reduction of proton gradient by nigericin or NH4Cl. pH measurements using fluorescent probes (acridine orange, quinacrine or enhanced green fluorescent protein–synaptobrevin) showed a strong correlation between vesicular pH and the kinetics of exocytosis. Hence, all maneuvers tested that decelerated exocytosis also alkalinized secretory vesicles and vice versa. On the other hand, calcium entry caused a transient acidification of granules. We therefore propose that the regulation of vesicular pH is, at least partially, a necessary step in the modulation of the kinetics of exocytosis and quantal size operated by some cell signals.

Intravesicular calcium release mediates the motion and exocytosis of secretory organelles. A study with adrenal chromaffin cells

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.

Chromogranin B Gene Ablation Reduces the Catecholamine Cargo and Decelerates Exocytosis in Chromaffin Secretory Vesicles

Chromogranins/secretogranins (Cgs) are the major soluble proteins of large dense-core secretory vesicles (LDCVs). We have recently reported that the absence of chromogranin A (CgA) caused important changes in the accumulation and in the exocytosis of catecholamines (CAs) using a CgA-knock-out (CgA-KO) mouse. Here, we have analyzed a CgB-KO mouse strain that can be maintained in homozygosis. These mice have 36% less adrenomedullary epinephrine when compared to Chgb+/+ [wild type (WT)], whereas the norepinephrine content was similar. The total evoked release of CA was 33% lower than WT mice. This decrease was not due to a lower frequency of exocytotic events but to less secretion per quantum (∼30%) measured by amperometry; amperometric spikes exhibited a slower ascending but a normal decaying phase. Cell incubation with l-DOPA increased the vesicle CA content of WT but not of the CgB-KO cells. Intracellular electrochemistry, using patch amperometry, showed that l-DOPA overload produced a significantly larger increase in cytosolic CAs in cells from the KO animals than chromaffin cells from the WT. These data indicate that the mechanisms for vesicular accumulation of CAs in the CgB-KO cells were saturated, while there was ample capacity for further accumulation in WT cells. Protein analysis of LDCVs showed the overexpression of CgA as well as other proteins apparently unrelated to the secretory process. We conclude that CgB, like CgA, is a highly efficient system directly involved in monoamine accumulation and in the kinetics of exocytosis from LDCVs.