Regulation by L channels of Ca2+-evoked secretory responses in ouabain-treated chromaffin cells

Abstract

It is known that the sustained depolarisation of adrenal medullary bovine chromaffin cells (BCCs) with high K(+) concentrations produces an initial sharp catecholamine release that subsequently fades off in spite depolarisation persists. Here, we have recreated a sustained depolarisation condition of BCCs by treating them with the Na(+)/K(+) ATPase blocker ouabain; in doing so, we searched experimental conditions that permitted the development of a sustained long-term catecholamine release response that could be relevant during prolonged stress. BCCs were perifused with nominal 0Ca(2+) solution, and secretion responses were elicited by intermittent application of short 2Ca(2+) pulses (Krebs-HEPES containing 2 mM Ca(2+)). These pulses elicited a biphasic secretory pattern with an initial 30-min period with secretory responses of increasing amplitude and a second 30-min period with steady-state, non-inactivating responses. The initial phase was not due to gradual depolarisation neither to gradual increases of the cytosolic calcium transients ([Ca(2+)]c) elicited by 2Ca(2+) pulses in BBCs exposed to ouabain; both parameters increased soon after ouabain addition. Νifedipine blocked these responses, and FPL64176 potentiated them, suggesting that they were triggered by Ca(2+) entry through non-inactivating L-type calcium channels. This was corroborated by nifedipine-evoked blockade of the L-type Ca(2+) channel current and the [Ca(2+)]c transients elicited by 2Ca(2+) pulses. Furthermore, the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) blocker SEA0400 caused a mild inhibition followed by a large rebound increase of the steady-state secretory responses. We conclude that these two phases of secretion are mostly contributed by Ca(2+) entry through L calcium channels, with a minor contribution of Ca(2+) entry through the reverse mode of the NCX.

Aluminium alters excitability by inhibiting calcium, sodium and potassium currents in bovine chromaffin cells

“…Adrenal glands were obtained from a local slaughterhouse under the supervision of the local veterinary service. Bovine chromaffin cells were isolated by digestion of the adrenal medulla with collagenase (de Pascual et al, 2016). Briefly, cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM), and were further supplemented with 5% foetal bovine serum, 50 IU/ml penicillin, in combination with 50 μg/ml streptomycin.…”

Novel Purine Derivative ITH15004 Facilitates Exocytosis through a Mitochondrial Calcium-Mediated Mechanism

“…BCCs were isolated from adrenal glands of adult cows following standard methods [ 28 ] with some modifications [ 29 , 30 ]. They were plated at a density of 5 × 10 6 cells per 5 mL of Dulbecco’s modified Eagle’s medium (DMEM) in 6 cm diameter Petri dishes (to study secretion), in 12 mm-diameter glass coverslips at a density of 5 × 10 4 cells (to study barium currents, I Ba ), or into 96-well black plates at a density of 2 × 10 5 cells per well, to monitor Ca 2+ signals.…”

“…To stimulate the catecholamine secretion, a Krebs–Hepes solution containing KCl at 35 mM with isosmotic reduction of NaCl (35K + solution), was applied in 5 s pulses at 1 min regular intervals at 37 ºC. After each stimulation, we measured in real time the catecholamine release by amperometry [ 30 , 31 , 32 ]. Representative records of some experiments shown in this report were accomplished by importing the data obtained in ASCII format to the Origin 8.6 (Microcal) program.…”

Tetrabenazine Facilitates Exocytosis by Enhancing Calcium-Induced Calcium Release through Ryanodine Receptors

“…All experiments were carried out in accordance with the guidelines established by the National Council on Animal Care and were approved by the local Animal Care Committee of the Universidad Autónoma de Madrid. BCCs were isolated from calf adrenal medullary tissues obtained from a local slaughterhouse, isolated according to Moro et al (1990) with some modifications (De Pascual et al, 2016), and plated at a density of 5 Â 10 6 cells per 5 ml of Dulbecco's modified Eagle's medium on 6-cmdiameter Petri dishes (to study secretion) and on 12-mm-diameter glass coverslips at a density of 5 Â 10 4 cells/coverslip to study calcium currents (I Ca ) through voltage-activated calcium channels (VACCs). Cells were kept for 1-4 days at 37°C in a water-saturated incubator in a 5% CO 2 /95% air atmosphere.…”

“…Both cells and perfusate were held at a temperature of 37°C. The liquid flowing from the superfusion chamber reached an electrochemical detector (model CH-9100; Metrohm AG, Herisau, Switzerland) placed just at the outlet of the microchamber, which monitors online the amount of catecholamines secreted under the amperometric mode (Green and Perlman, 1981;Borges et al, 1986;De Pascual et al, 2016). The catecholamines present in the perfusate were oxidized at 10.65 V, and the oxidation current was recorded with a frequency of 2 Hz to monitor the amount of total catecholamine secreted (Borges et al, 1986).…”