Calcium currents in the A7r5 smooth muscle-derived cell line

Area of Science:

• Pharmacology
• Cell Biology
• Physiology

Background:

• A7r5 smooth muscle cells are a common model for studying cellular responses.
• Voltage-dependent calcium channels play a crucial role in smooth muscle contraction and cellular signaling.
• Dihydropyridines (DHPs) are a class of drugs known to interact with L-type calcium channels.

Purpose of the Study:

• To characterize the voltage-dependent calcium channels in the A7r5 smooth muscle cell line.
• To investigate the role of these channels in regulating cytosolic calcium levels and cellular activity.
• To determine the pharmacological properties of DHP binding sites in these cells.

Main Methods:

• Radiolabelled dihydropyridine binding assays.
• Whole-cell and single-channel patch-clamp electrophysiology.
• Measurement of cytosolic calcium ([Ca2+]i) using fura-2.
• Voltage-clamp experiments with barium as the charge carrier.
• Pharmacological manipulation with DHPs, isoproterenol, forskolin, Bay K 8644, and nifedipine.

Main Results:

• A7r5 cells exhibit saturable, high-affinity, voltage-sensitive DHP binding sites characteristic of L-type calcium channels.
• A dominant DHP-sensitive inward current, resembling L-type calcium current, was observed.
• Peak inward currents occurred at 0 to +20 mV and were blocked by cadmium (IC50 ≈ 1 µM).
• Isoproterenol or forskolin modulated currents in about half of the cells, and forskolin increased single-channel activity.
• Cell suspensions showed altered resting [Ca2+]i in response to DHPs and potassium.
• Confluent monolayers displayed spontaneous cytosolic calcium ([Ca2+]i) transients, enhanced by Bay K 8644 and abolished by nifedipine.

Conclusions:

• A7r5 cells express functional L-type calcium channels.
• These channels are responsible for mediating cytosolic calcium ([Ca2+]i) transients.
• The observed [Ca2+]i transients in monolayers are likely due to synchronous action potentials in electrically coupled cells.