M

M. gland, the main source of adrenaline under nerve-racking situations.Prez-Alvarez, A., Hernndez-Vivanco, A., McIntosh, J. M., Albillos, A. Native 64* nicotinic receptors control exocytosis in human chromaffin cells of the adrenal gland. in 1990 (12). Since then, transcripts of 6 nAChRs have been shown to be largely expressed presynaptically in catecholaminergic neurons of the central nervous system (8, 13, 14). Recently, functional nAChRs made up of 6 subunits have been reported in GABAergic neuronal boutons adherent to ventral 3-Methylcytidine tegmental area dopamine neurons (15). The presence of 6 nAChR subunits in the chromaffin cells of the human adrenal gland medulla further supports the idea that 6* nAChRs are mainly expressed in catecholaminergic cells. 6* nAChRs control release of dopamine (16, 17), noradrenaline (18, 19), and GABA (15). Their expression and function are affected by chronic exposure to nicotine (20C22). These receptors also show a role in nicotine incentive and affective nicotine withdrawal (23). In addition, there is selective loss of 6 nAChRs in Parkinson’s disease (22, 24, 25). In the present study, we show that a populace of nAChRs expressed in the chromaffin cells of the human adrenal medulla are composed of 6 and 4 nAChR subunits and that these 64* nAChRs mostly control the exocytotic process. Because chromaffin cells of the adrenal gland constitute the major source of adrenaline under stress situations, the therapeutic regulation of 64* nAChRs might have crucial clinical effects. MATERIALS AND METHODS Cell cultures The study protocol was approved by the ethics committees of the Hospital Ramn y Cajal (Madrid, Spain) and Universidad Autnoma de Madrid. After informed consent was obtained from the donors’ relatives, adrenal glands were harvested from 9 adult organ donors who experienced died of cerebral hemorrhage. The inherent troubles in obtaining human adrenal glands limited the number of experiments that could be performed. The method utilized for isolation and culture of human chromaffin cells has been explained previously (1). Experiments were started 48 h after plating to allow recovery of the nicotinic receptor after collagenase treatment (26). The protocol for mouse and rat chromaffin cell cultures was performed as explained previously (27). Electrophysiological recordings Perforated patch recordings were made in the whole-cell configuration of the patch-clamp technique. The external solution used to record nicotinic currents was 2 mM CaCl2, 145 mM NaCl, 5.5 mM KCl, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose; pH was adjusted to 7.4 with NaOH. Intracellular answer composition was 145 mM potassium glutamate, 8 mM NaCl, 1 mM MgCl2, 10 mM HEPES, and 3-Methylcytidine 0.5 mM amphotericin B (Sigma-Aldrich, Madrid, Spain); pH was adjusted to 7.2 with KOH. An amphotericin B stock solution was prepared daily at a concentration of 50 mg/ml DMSO and kept guarded from light. The final concentration of amphotericin B was prepared by ultrasonication of 10 l of stock amphotericin B in 1 ml of internal solution in the dark. Pipettes were tip-dipped in amphotericin-free answer for several seconds and back-filled with freshly mixed intracellular amphotericin answer. The perfusion system for drug application consisted of a multibarrelled polyethylene pipette situated close to the cell under study. The.Goldner F. Alexa Fluor 546-BuIA showed unique staining in these cells. Our results reveal that 64* nAChRs are expressed and contribute to exocytosis in human chromaffin cells of the adrenal gland, the main source of adrenaline under nerve-racking situations.Prez-Alvarez, A., Hernndez-Vivanco, A., McIntosh, J. M., Albillos, A. Native 64* nicotinic receptors control exocytosis in human chromaffin cells of the adrenal gland. in 1990 (12). Since then, transcripts of 6 nAChRs have been shown to be largely expressed presynaptically in catecholaminergic neurons of the central nervous system (8, 13, 14). Recently, functional nAChRs made up of 6 subunits have been reported in GABAergic neuronal boutons adherent to ventral tegmental area dopamine neurons (15). The presence of 6 nAChR subunits in the chromaffin cells of the human adrenal gland medulla further supports the idea that 6* nAChRs are mainly expressed in catecholaminergic cells. 6* nAChRs control release of dopamine (16, 17), noradrenaline (18, 19), and GABA (15). Their expression and function are affected by chronic exposure to nicotine (20C22). These receptors also show a role in nicotine incentive and affective nicotine withdrawal (23). In addition, there is selective loss of 6 nAChRs in Parkinson’s disease (22, 24, 25). In the present study, we show that a populace of nAChRs expressed in the chromaffin cells of the human adrenal medulla are composed of 6 and 4 nAChR subunits and that these 64* nAChRs mostly control the exocytotic process. Because chromaffin cells of the adrenal gland constitute the major source of adrenaline under stress situations, the therapeutic regulation of 64* nAChRs might have crucial clinical consequences. MATERIALS AND METHODS Cell cultures The study protocol was approved by the ethics committees of the Hospital Ramn y Cajal (Madrid, Spain) and Universidad Autnoma de Madrid. After informed consent was obtained from the donors’ relatives, adrenal glands were harvested from 9 adult organ donors who experienced died of cerebral hemorrhage. The inherent troubles in obtaining human adrenal glands limited the number of experiments that could be performed. The method utilized for isolation and culture of human chromaffin cells has been explained previously (1). Experiments were started 48 h after plating to allow recovery of the nicotinic receptor after collagenase treatment (26). The protocol for mouse and rat chromaffin cell cultures was performed as explained previously (27). Electrophysiological recordings Perforated patch recordings were made in the whole-cell configuration of the patch-clamp technique. The external solution used to record nicotinic currents was 2 mM CaCl2, 145 mM NaCl, 5.5 mM KCl, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose; pH was adjusted to 7.4 with NaOH. Intracellular answer composition was 145 mM potassium glutamate, 8 mM NaCl, 1 mM MgCl2, 10 mM HEPES, and 0.5 mM amphotericin B (Sigma-Aldrich, Madrid, Spain); pH was adjusted to 7.2 with KOH. An amphotericin B stock solution was prepared daily at a PLA2G4C concentration of 50 mg/ml DMSO and kept guarded from light. The final concentration of amphotericin B was prepared by ultrasonication of 10 l of stock amphotericin B in 1 ml of internal solution in the dark. Pipettes were tip-dipped in amphotericin-free answer for several 3-Methylcytidine seconds and back-filled with freshly mixed intracellular amphotericin answer. The perfusion system for drug application consisted of a multibarrelled polyethylene pipette positioned close to the cell under study. The exchange time of solutions of this system calculated with open-tip experiments (28) was 10 ms. The agonist was always delivered from the same tube. Antagonists were perfused between pulses, and this flow was only interrupted during agonist perfusion (200 ms). Time between pulses was 2 or 5 min. The level of the bath fluid was continuously controlled by a custom-designed fiberoptics system coupled to a pump used to aspirate excess fluid. Pipettes of 2C3 M resistance were pulled from borosilicate glass capillary tubes, partially coated with wax, and fire-polished. After seal formation and perforation, only recordings in which the access resistance of the pipette and the leak current were lower than 20 M and 20 pA, respectively, were accepted. The holding potential (tests were used to compare data. Fluorescent staining of live human chromaffin cells A protocol similar to that used by Hone (36) was used to stain.