Related Experiment Videos
Modulation of N-type calcium channels translocation in RINm5F insulinoma cells
M Passafaro1, A Codignola, M Rogers
1Department of Medical Pharmacology, CNR Molecular and Cellular Pharmacology Center, University of Milan, Milan, 20129, Italy.
Abstract:
An intracellular pool of N-type voltage-operated calcium channels has recently been described in both IMR32 human neuroblastoma and PC12 rat pheochromocytoma cells. These channels were found to be accumulated in subcellular fractions where the chromogranin B-containing secretory granules were also enriched. Upon exocytosis N-type calcium channels were reversibly inserted in the plasma membrane. We have now extended this study to RINm5F rat insulinoma cells, and characterized the parallelism between the 'regulated' secretion of serotonin and the recruitment of surface calcium channels. Exocytosis was stimulated by different means, such as depolarization with high KCl, high Ba(2+)alone or protein kinase C activation; on the other hand exocytosis was inhibited with the non-selective calcium channel antagonist Cd(2+)or with noradrenaline. Stimulated release was always accompanied, with parallel kinetics, by calcium channel recruitment, while inhibition of secretion blocked calcium channel recruitment too. During repetitive depolarizations we revealed a potentiation of [Ca(2+)]()i transients in single Fura-2 loaded RINm5F cells, that was accompanied by an increase in surface VOCCs, suggesting a physiological role for the newly recruited channels. 2000 Academic Press@p$hr
Insights
N-type voltage-operated calcium channels are stored intracellularly and insert into the plasma membrane during regulated exocytosis in RINm5F cells. This recruitment parallels serotonin secretion, suggesting a role in calcium signaling.
Area of Science:
- Cell Biology
- Neuroscience
- Endocrinology
Background:
- N-type voltage-operated calcium channels (VOCCs) have an intracellular pool.
- These channels accumulate with secretory granules in neuroblastoma and pheochromocytoma cells.
- Upon exocytosis, N-type VOCCs are inserted into the plasma membrane.
Purpose of the Study:
- To investigate the relationship between regulated exocytosis and N-type VOCC recruitment in RINm5F rat insulinoma cells.
- To characterize the kinetics of VOCC recruitment during stimulated and inhibited exocytosis.
- To explore the physiological role of recruited VOCCs in calcium transients.
Main Methods:
- Stimulation of exocytosis using high KCl, high Ba(2+), and protein kinase C activation.
- Inhibition of exocytosis using cadmium (Cd(2+)) and noradrenaline.
- Monitoring of serotonin secretion and surface VOCCs.
- Measurement of intracellular calcium ([Ca(2+)]i) transients in single Fura-2 loaded cells during repetitive depolarizations.
Main Results:
- Exocytosis stimulation led to parallel recruitment of surface calcium channels.
- Inhibition of exocytosis blocked calcium channel recruitment.
- Repetitive depolarizations showed potentiation of intracellular calcium transients.
- This potentiation was associated with an increase in surface VOCCs.
Conclusions:
- Regulated exocytosis in RINm5F cells is tightly coupled to the recruitment of intracellular N-type VOCCs to the plasma membrane.
- Recruited VOCCs likely play a physiological role in enhancing calcium influx during sustained stimulation.
- This mechanism highlights a novel pathway for regulating calcium signaling in endocrine cells.