Key role of the nicotinic receptor in neurotransmitter exocytosis in human chromaffin cells.
The whole-cell secretory response evoked by acetylcholine (ACh) in human chromaffin cells was examined using a new protocol based on quickly switching from the voltage-clamp to the current-clamp (CC) configuration of the patch-clamp technique. Our experiments revealed that Ca(2+) entry through the nicotinic receptor at hyperpolarized membrane potentials contributed as much to the exocytosis (100.4 +/- 27.3 fF) evoked by 200 ms pulses of ACh, as Ca(2+) flux through voltage-dependent Ca(2+) channels at depolarized membrane potentials. The nicotinic current triggered a depolarization event with a peak at +49.3 mV and a 'plateau' phase that ended at -23.9 mV, which was blocked by 10 mumol/L mecamylamine. When a long ACh stimulus (15 s) was applied, the nicotinic current at the end of the pulse reached a value of 15.45 +/- 3.6 pA, but the membrane potential depolarization still remained at the 'plateau' stage until withdrawal of the agonist. Perfusion with 200 mumol/L Cd(2+) during the 15 s ACh pulse completely abolished the plasma membrane depolarization at the end of the pulse, indicating that Ca(2+) entry through Ca(2+) channels contributed to the membrane potential depolarization provoked by prolonged ACh pulses. These findings also reflect that voltage-dependent Ca(2+) channels were recruited by the small current flowing through the desensitized nicotinic receptor to maintain the depolarization. Finally, muscarinic receptor activation triggered a delayed exocytotic process after prolonged ACh stimulation, dependent on Ca(2+) mobilization from the endoplasmic reticulum. In summary, we show here that nicotinic and muscarinic receptors contribute to the exocytosis of neurotransmitters in human chromaffin cells, and that the nicotinic receptor plays a key role in several stages of the stimulus-secretion coupling process in these cells.
Pérez-Alvarez
Alberto
A
Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain.
Albillos
Almudena
A
D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology; D000313:Adrenal Medulla / Q000166:cytology / Q000378:metabolism*; D000328:Adult; D000368:Aged; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005260:Female; D006801:Humans; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008875:Middle Aged; D018377:Neurotransmitter Agents / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology*
D018377:Neurotransmitter Agents; D011976:Receptors, Muscarinic; D011978:Receptors, Nicotinic; D000109:Acetylcholine