Related Experiment Videos
Methylmercury blocks N- and L-type Ca++ channels in nerve growth factor-differentiated pheochromocytoma (PC12) cells
1Department of Pharmacology and Toxicology, Michigan State University, East Lansing.
Abstract:
Effects of methylmercury (MeHg) on whole-cell Ba++ currents in rat pheochromocytoma (PC12) cells were examined. Based on biophysical characteristics and sensitivity to omega-conotoxin GVIA and dihydropyridine agonists and antagonists, voltage-activated Ba++ currents (IBa) in PC12 cells were mediated by N- and L-type Ca++ channels. Addition of MeHg (10 microM) to the extracellular solution caused a rapid and complete block of current carried by 20 mM Ba++. The rate of block of IBa by MeHg increased in a concentration-dependent manner between 1 and 20 microM. Increasing the frequency of stimulation from 0.1 to 0.4 Hz facilitated block of IBa by MeHg. A 2-min application of 10 microM MeHg in the absence of stimulation also reduced IBa by approximately 80%. Thus, block of IBa by MeHg is not state-dependent. Additionally, MeHg blocked IBa when the membrane holding potential was -40, -70 and -90 mV, indicating that both N- and L-type Ca++ channels are blocked by MeHg. Block of IBa by MeHg was voltage-dependent at a membrane holding potential of -40 mV, but not at holding potentials of -70 and -90 mV. Decreasing the extracellular concentration of Ba++ ([Ba++]e) from 20 mM to 10 mM increased the magnitude of block by MeHg from 45.6 to 77.3%. Increasing [Ba++]e to 30 mM caused no further antagonism of block. Block of IBa by MeHg was not reversed by washing with MeHg-free solution. The ionic permeability of PC12 cell Ca++ channels was Ca++ = Sr++ greater than Ba++. In the presence of MeHg, all three divalent cations were equally permeant.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Methylmercury (MeHg) rapidly and completely blocks barium currents (IBa) in rat PC12 cells by inhibiting both N- and L-type calcium channels. This block is concentration-dependent, voltage-dependent, and not reversed by washing, indicating significant neurotoxicity.
Area of Science:
- Neuropharmacology
- Toxicology
- Cell Physiology
Background:
- Rat pheochromocytoma (PC12) cells possess voltage-activated barium currents (IBa) mediated by N- and L-type calcium channels.
- Methylmercury (MeHg) is a known neurotoxin, but its specific effects on calcium channel subtypes require detailed investigation.
Purpose of the Study:
- To investigate the effects of methylmercury (MeHg) on whole-cell barium currents (IBa) in rat pheochromocytoma (PC12) cells.
- To determine the specific calcium channel subtypes affected by MeHg and characterize the nature of the block.
Main Methods:
- Whole-cell patch-clamp electrophysiology was used to record voltage-activated Ba++ currents (IBa) in PC12 cells.
- Cells were exposed to varying concentrations of MeHg, stimulation frequencies, and holding potentials.
- The effects of MeHg on Ba++ currents were analyzed, including concentration-dependence, frequency-dependence, and voltage-dependence.
Main Results:
- MeHg (10 microM) caused rapid and complete block of IBa, with the rate of block increasing concentration-dependently (1-20 microM).
- MeHg blocked IBa independently of channel state and affected both N- and L-type calcium channels, with voltage-dependent block observed at -40 mV.
- Decreasing extracellular Ba++ concentration enhanced MeHg block, and the block was irreversible upon washing.
Conclusions:
- Methylmercury potently inhibits both N- and L-type calcium channels in PC12 cells.
- MeHg-induced channel block is concentration-dependent, partially voltage-dependent, and irreversible, suggesting significant neurotoxic potential.
- MeHg alters the ionic permeability of calcium channels, making Ca++, Sr++, and Ba++ equally permeant in its presence.