Differential variations in Ca2+ entry, cytosolic Ca2+ and membrane capacitance upon steady or action potential depolarizing stimulation of bovine chromaffin cells.
OBJECTIVE
This study looks into the physiology of the exocytosis of catecholamines released by adrenal medullary chromaffin cells. We have comparatively explored the exocytotic responses elicited by two different patterns of depolarizing stimulation: the widely employed square depolarizing pulses (DPs) and trains of acetylcholine-like action potentials (APs), likely the physiological mode of stimulation in the intact innervated adrenal medulla. APs were applied at 30 Hz, a frequency similar to that produced in a stressful situation.


METHODS
Patch-clamp, cell membrane capacitance, single cell amperometry and fluorescence were the techniques used. The variations of calcium entry measured as the integral of the calcium current, cytosolic calcium (measured with the calcium-sensitive fluorescent probe fluo-4) and exo-endocytosis (membrane capacitance variations) were the parameters measured.


RESULTS
Trains of AP depolarizations produced distinct responses compared to those of square depolarizations: (1) Calcium current amplitude decreased to a lesser extent along the AP train; (2) calcium entry and capacitance increments raised linearly with stimulation time whereas they deviated from linearity when square depolarizations were used; (3) slower activation and faster delayed decay phase of cytosolic calcium transients; (4) capacitance increments varied linearly with calcium entry with APs and deviated from linearity with longer depolarizations; (5) little endocytosis after stimulation with longer trains of APs and pronounced endocytosis with longer square depolarizations.


CONCLUSIONS
Stimulation of chromaffin cells with trains of APs produced patterns of cytosolic calcium transients, exocytotic and endocytotic responses quite different from those elicited by the widely employed DPs. Our study is relevant from the methodological and physiological points of view.
de Diego
A M G
AM
Facultad de Medicina, Instituto Teófilo Hernando, Universidad Autónoma de Madrid, Madrid, Spain. antoniomiguel.garcia@uam.es
Arnáiz-Cot
J J
JJ
Hernández-Guijo
J M
JM
Gandía
L
L
García
A G
AG
D000109:Acetylcholine / Q000494:pharmacology; D000200:Action Potentials / Q000187:drug effects / Q000502:physiology*; D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D016764:Cell Polarity / Q000502:physiology; D002478:Cells, Cultured; D018678:Cholinergic Agents / Q000494:pharmacology; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D003600:Cytosol / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods
D018678:Cholinergic Agents; D000109:Acetylcholine; D002118:Calcium