Calcium dynamics in catecholamine-containing secretory vesicles

Cellular and Molecular Neurobiology, 2010

We have investigated the dynamics of the free [Ca 2? ] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca 2?-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca 2? ] ([Ca 2? ] SG ] was around 20-40 lM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca 2? pumps with thapsigargin largely blocked Ca 2? uptake by the granules in Ca 2?-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca 2? pump inhibitor benzohydroquinone induced a rapid release of Ca 2? from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca 2? pumps is essential to maintain the steady-state [Ca 2? ] SG. Both inositol 1,4, 5-trisphosphate (InsP 3) and caffeine produced a rapid Ca 2? release from the granules, suggesting the presence of InsP 3 and ryanodine receptors in the granules. The response to high-K ? depolarization was different in both cell types, a decrease in [Ca 2? ] SG in PC12 cells and an increase in [Ca 2? ] SG in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca 2? ] SG in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca 2? through SERCA-type Ca 2? pumps and can release it through InsP 3 and ryanodine receptors, supporting the hypothesis that secretory granule Ca 2? may be released during cell stimulation and contribute to secretion. Keywords Ca 2? Á Secretory granules Á PC12 cells Á INS1 cells Á Inositol 1,4,5-trisphosphate receptors Á Ryanodine receptors Á Aequorin Abbreviations BHQ tert-Butyl benzohydroquinone [Ca 2? ] SG Secretory granule [Ca 2? ] DMPP 1,1-Dimethyl-4-phenyl-piperazinium iodide InsP 3 Inositol 1,4,5-trisphosphate SERCA Sarco endoplasmic reticulum Ca 2? ATPase VAMP Vesicle-associated membrane protein VAMP-mutaeq VAMP-mutated aequorin chimera

European Journal of Pharmacology: Molecular Pharmacology, 1991

A number of commonly used calcium channel blockers have been compared with respect to their effects on the bioenergetics of catecholamine storage vesicles~ Chromaffin granule ghosts with a well-preserved ability to actively transport and store catecholamines, were used as a model for adrenergic synaptic vesicles due to their functional similarity. Nicardipine, verapamil, terodiline and diltiazem were found to have effects comparable to that of prenylamine (Gronberg, M., O. Terland, E.S. Husebye and T. Flatmark, 1990, Biochem. Pharmacol. 40, 351) by inhibiting the generation of a transmembrane proton electrochemical gradient driven by the vesicular H÷-ATPase, mainly by loose-coupling/uncoupling of this ATPase. Amlodipine inhibited the internal acidification of the vesicles in a tyramine-like manner and increased the steady-state membrane potential (positive inside) generated by the MgATP-dependent proton translocation. Nifedipine and felodipine also inhibited the efficiency of the proton pump, but their mechanisms of action require further investigation. The concentrations giving 50% inhibition of the H +-ATPasedependent generation of a pH-gradient were found to be: 12 #M felodipine, 16 #M nicardipine, 25 #M terodiline, 50 #M nifedipine, 60 #M verapamil, 65 /zM amlodipine and 150 #M diltiazem. The effects of the calcium channel blockers on the bioenergetics of chromaffin granules explain the release of catecholamines from sympathetic nerves and ganglia in vitro by the calcium channel blockers.

Biochemical Pharmacology, 2000

Annals of The New York Academy of Sciences, 2002

Abstract: At a given cytosolic domain of a chromaffin cell, the rate and amplitude of the Ca2+ concentration, [Ca2+]c, depend on at least three efficient regulatory mechanisms: (1) the plasmalemmal Ca2+ channels; (2) the endoplasmic reticulum (ER); and (3) the mitochondria. High-voltage activated Ca2+ channels of the L, N, P/Q, and R subtypes are expressed with different densities in various mammalian species; they are regulated by G proteins coupled to purinergic and opiate receptors, as well as by voltage and the local changes of [Ca2+]c. Targeted aequorin and confocal microscopy show that Ca2+ entry through Ca2+ channels can refill the ER to near millimolar concentrations and causes the release of ER Ca2+ (CICR). We have also seen that, depending on its degree of filling, the ER may act as a sink or source of Ca2+ that modulates the release of catecholamine. Targeted aequorins with different Ca2+ affinities show that mitochondria undergo surprisingly rapid millimolar Ca2+ transients ([Ca2+]M) upon stimulation of chromaffin cells with ACh, high K+, or caffeine. Physiological stimuli generate [Ca2+]c microdomains at these functional complexes in which the local subplasmalemmal [Ca2+]c rises abruptly from 0.1 μM to about 50 μM. This triggers CICR, mitochondrial Ca2+ uptake, and exocytosis in nearby secretory active sites. That this is true is shown by the observation that protonophores abolish mitochondrial Ca2+ uptake and drastically increase catecholamine release by 3- to 5-fold. This increase is likely due to acceleration of vesicle transport from a reserve pool to a ready-release vesicle pool; such transport might be controlled by Ca2+ redistribution to the cytoskeleton, through CICR and/or mitochondrial Ca2+ release.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1987

The (Ca2+ + MgZ+)-ATPase was purified from skeletal muscle sarcoplasmic reticulum and reconstituted into sealed phospholipid vesicles by solution in cholate and deoxycholate followed by detergent removal on a column of Seplmdex G-50. The level of Ca 2+ accumulated by these vesicles, either in the presence or absence of phosphate within the vesicles, increased with increasing content of phosphatidylethanolamine in the phospholipid mixture used for the reconstitution. The levels of Ca 2+ accumulated in the absence of phosphate were very low for vesicles reconstituted with egg yolk phosphatidyicholine alone at pH 7.4, but increased markedly with decreasing pH to 6.0. Uptake was also relatively low for vesicles recoltstituted with dimyristoleoyl-or dinervonylphosphatidylcholine, and addition of cholesterol had little effect. The level of Ca 2+ accumulated increased with increasing external K + concentration, and was also increased by the ionophores FCCP and valinomycin. Vesicle sizes changed little with changing phosphatidylethanolamine content, and the sidedness of insertion of the ATPase was close to random at all phosphatidylethanolamine contents. It is suggested that the effect of phosphatidylethanolamine on the level of Ca 2+ accumulation follows from an effect on the rate of Ca 2+ efflux mediated by the ATPase.

Journal of Biological Chemistry, 1996

Parafollicular (PF) cells secrete 5-hydroxytryptamine in response to increased extracellular Ca 2؉ (1[Ca 2؉ ] e). This stimulus causes Cl ؊ channels in PF secretory vesicles to open, leading to vesicle acidification. PF cells express a plasmalemmal heptahelical receptor (CaR) that binds Ca 2؉ , Gd 3؉ , and Ba 2؉. We now report that the CaR mediates vesicle acidification. Ca 2؉ , Gd 3؉ , and Ba 2؉ induced vesicle acidification, which was independent of channel-mediated Ca 2؉ entry. Agonist-induced vesicle acidification was blocked by pertussis toxin, inhibitors of phosphatidylinositol-phospholipase C, calmodulin, NO synthase, guanylyl cyclase, or protein kinase G. PF cells contained NO synthase immunoreactivity, and vesicles were acidified by NO donors and dibutyryl cGMP. [Ca 2؉ ] e , and Gd 3؉ mobilized thapsigargin-sensitive internal Ca 2؉ stores. [ 35 S]G ␣i and [ 35 S]G ␣q were immunoprecipitated from PF membranes incubated with agonists in the presence of [ 35 S]adenosine 5-O-(thiotriphosphate). Labeling of G ␣i but not G ␣q was antagonized by pertussis toxin. Vesicles acidified in response to activation of protein kinase C; however, protein kinase C inhibition blocked calcium channel-but not CaR-dependent acidification. We propose the following signal transduction pathway: CaR 3 G i 3 phosphatidylinositol-phospholipase C 3 inositol 1,4,5-trisphosphate 3 1[Ca 2؉ ] i 3 Ca 2؉ /calmodulin 3 NO synthase 3 NO 3 guanylyl cyclase 3 cGMP 3 protein kinase G 3 opens vesicular Cl ؊ channel.

Journal of Biological Chemistry

Release of Ca2+ from intracellular stores was studied in the parent PC12 cell line and in recently isolated clones sensitive or insensitive to caffeine. In the caffeine-sensitive cells the cytosolic free Ca2+ concentration ([Ca2+]i) responses by the xanthine drug and by stimulants of receptors coupled to inositol 1,4,5-trisphosphate (Ins-P3) generation (bradykinin, ATP) depend on separate pathways because 1) caffeine does not stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate and 2) Ca(2+)-induced Ca2+ release, the process activated by caffeine, plays no major role in the Ins-P3-induced Ca2+ mobilization. Although distinct, these two mechanisms converge onto the same Ca2+ store. In fact 1) the [Ca2+]i responses by receptor agonists and caffeine were not additive; 2) either type of agent reduced (up to complete inhibition) the response to a subsequent administration of the same or the other agent; 3) all these responses were prevented by selective Ca2+ ATPase blockers;...

The Journal of Membrane Biology, 2011

Translocation of vesicles within the cytoplasm is essential to normal cell function. The vesicles are typically transported along the microtubules to their destination. The aim of this study was to characterize the vesicular movement in resting and stimulated renal epithelial cells. MDCK cells loaded with either quinacrine or acridine orange, dyes taken up by acidic vesicles, were observed at 37°C in semiopen perfusion chambers. Time-lapse series were analyzed by Imaris software. Our data revealed vigorous movement of stained vesicles in resting MDCK cells. These movements seem to require intact microtubules because nocodazole leads to a considerable reduction of the vesicular movements. Interestingly, we found that extracellular ATP caused the vesicular movement to cease. This observation was obvious in time lapse. Similarly, other stimuli known to increase the intracellular Ca 2? concentration ([Ca 2? ] i ) in MDCK cells (increment in the fluid flow rate or arginine vasopressin) also reduced the vesicular movement. These findings were quantified by analysis of single vesicular movement patterns. In this way, ATP was found to reduce the lateral displacement of the total population of vesicles by 40%. Because all these perturbations increase [Ca 2? ] i , we speculated that this increase in [Ca 2? ] i was responsible for the vesicle arrest. Therefore, we tested the effect of the Ca 2? ionophore, ionomycin (1 lM), which in the presence of extracellular Ca 2? resulted in a considerable and sustained reduction of vesicular movement amounting to a 58% decrease in average lateral vesicular displacement. Our data suggest that vesicles transported on microtubules are paused when subjected to high intracellular Ca 2? concentrations. This may provide an additional explanation for the cytotoxic effect of high [Ca 2? ] i .

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988

An ATP-driven Ca z+ pump in the Imsolateral membrane of rat kidney cortex pumps Ca z+ out of the cell at the expense of MgATP (K m = 0.191 raM). TMs pump has a high affinity for free Ca ~+ (26 nM). Vanadate, lanthanum, N-ethylmleimide and calmoduUn inldhitor 1124571 inhibited this pump activity. Dimethyi12-UC]oxazolidine.2,4-dione ([uC]DMO) was entrapped in the vesicles in association with the ATP-driven Ca 2+ influx. The ATP-ddven Ca z+ influx was stimulated by the intravesicniar acid pH and an upper convex Lineweaver-Burk reciprocal plot suggested two possible kinetics; one is that this Ca z~" pump is an allosteric enzyme with more than 1.72 H + binding sites and ~mother is the presence of two Ca z + pumps with different affinities for H +. Valinomycin study indicated that the ATP-dependent Ca z ÷ transport by the BLMV was eleetroneutrul and voltage independent. These results strongly suggest that tl~. ATP.driven Ca z+ pump in the renal he~olaterai membrane catalyzes an eleetroneutral Ca2+/H + antipo~

Journal of Biological Chemistry, 2004

Chromaffin vesicles contain very high concentration of Ca 2؉ (ϳ20-40 mM total), compared with ϳ100 nM in the cytosol. Aequorin, a jellyfish photoprotein with Ca 2؉-dependent luminescence, measures [Ca 2؉ ] in specific subcellular compartments wherein proteins with organellespecific trafficking domains are fused in-frame to aequorin. Because of the presence of vesicular trafficking domain within CgA we engineered sorting of an expressed human CgA-Aequorin fusion protein (hCgA-Aeq) into the vesicle compartment as confirmed by sucrose density gradients and confocal immunofluorescent co-localization studies. hCgA-Aeq and cytoplasmic aequorin (Cyto-Aeq) luminescence displayed linear functions of [Ca 2؉ ] in vitro, over >5 log 10 orders of magnitude (r > 0.99), and down to at least 10 ؊7 M sensitivity. Calibrating the pH dependence of hCgA-Aeq luminescence allowed estimation of [Ca 2؉ ] ves at granule interior pH (ϳ5.5). In the cytoplasm, Cyto-Aeq accurately determined [Ca 2؉ ] cyto under both basal ([Ca 2؉ ] cyto ‫؍‬ 130 ؎ 35 nM) and exocytosis-stimulated conditions, confirmed by an independent reference technique (Indo-1 fluorescence). The hCgA-Aeq chimera determined vesicular free [Ca 2؉ ] ves ‫؍‬ 1.4 ؎ 0.3 M under basal conditions indicating that >99% of granule total Ca 2؉ is in a "bound" state. The basal free [Ca 2؉ ] ves /[Ca 2؉ ] cyto ratio was thus ϳ10.8-fold, indicating active, dynamic Ca 2؉ uptake from cytosol into the granules. Stimulation of exocytotic secretion revealed prompt, dynamic increases in both [Ca 2؉ ] ves and [Ca 2؉ ] cyto , and an exponential relation between the two (y ‫؍‬ 0.99 ؋ e (1.53x) , r ‫؍‬ 0.99), reflecting a persistent [Ca 2؉ ] ves /[Ca 2؉ ] cyto gradient, even during sharp increments of both values. Studies with inhibitors of Ca 2؉ translocation (Ca 2؉-ATPase), Na ؉ /Ca ؉-exchange, Na ؉ /H ؉exchange, and vesicle acidification (H ؉-translocating ATPase), documented a role for these four ion transporter classes in accumulation of Ca 2؉ inside the vesicles.