Confocal calcium imaging reveals an ionotropic P2 nucleotide receptor in the paranodal membrane of rat Schwann cells

Abstract

1. The paranodal Schwann cell region is of major importance for the function of a myelinated axon. In the present study we searched for a possible ionotropic effect of extracellular ATP in this Schwann cell compartment. 2. Whole-cell patch-clamp recordings from cultured rat Schwann cells revealed that ATP and 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP) induced a non-specific cation current. The effect of ATP was much enhanced in a Ca2+- and Mg2+-free solution. ADP, UTP and alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP) had no effect. 3. Confocal Ca2+ imaging of myelinating Schwann cells in isolated rat spinal roots showed a BzATP-induced rise in the free intracellular Ca2+ concentration in the paranodal Schwann cell cytoplasm whereas alpha,beta-meATP and 2-(methylthio)-adenosine 5'-triphosphate were without effect. In contrast to the known metabotropic effect of UTP on these Schwann cell regions, the BzATP-induced Ca2+ signal was not transient, was unaffected by depletion of intracellular Ca2+ stores and dependent on the presence of extracellular Ca2+. 4. These results suggest that an ionotropic ATP receptor with electrophysiological and pharmacological characteristics of the P2X7 subtype of nucleotide receptors is functionally active in myelinating Schwann cells of peripheral nerves. Such a receptor might contribute to Schwann cell reactions in nerve injury or neuropathy.

Figure 1. ATP and BzATP induce membrane currents in cultured rat Schwann cells

The illustrated examples from four different Schwann cells are representative for the effects of ATP and BzATP. A and B, membrane currents induced by slow voltage ramps from -140 to 80 mV in 2 s were recorded from two different cultured rat Schwann cells before and during bath application of ATP (1 mM) and BzATP (150 μM). Note the different scaling of the current axes. C and D, the membranes of these Schwann cells were voltage clamped at a constant holding potential (E_m) and changes in the holding current were recorded during bath application of different P2 nucleotide receptor agonists.

Figure 2. BzATP induces a rise in [Ca²⁺]_i in the paranodal Schwann cell cytoplasm

A, confocal image of myelinated nerve fibres within an isolated rat spinal root stained with the Ca²⁺-sensitive dyes Calcium Green-1 and Fura Red (excitation wavelength 488 nm; emission wavelengths 530 and 660 nm, respectively). B and C, the mean grey value of the paranodal Schwann cell area (selected region of interest indicated in A) and its normalized ratio was measured during bath application of BzATP (150 μM; B) and UTP (300 μM; C). Note the maintained rise in [Ca²⁺]_i during application of BzATP whereas UTP induced a transient rise in [Ca²⁺]_i only.

Figure 3. BzATP induces a transmembrane Ca²⁺ influx

A, the white outline in the upper panel shows a paranodal Schwann cell region within an isolated rat spinal root stained with the Ca²⁺-sensitive dyes Calcium Green-1 and Fura Red. Changes in [Ca²⁺]_i were measured during application of BzATP (150 μM) into the standard and into a Ca²⁺-free bathing solution. B, a similar experimental protocol was applied to a different rat spinal root. In this case, changes in [Ca²⁺]_i were measured during application of UTP (300 μM) into the standard and into a Ca²⁺-free bathing solution. Note that only the BzATP-induced intracellular Ca²⁺ transient was completely and reversibly blocked during removal of extracellular Ca²⁺.

Figure 4. BzATP does not release Ca²⁺ from intracellular stores

A, confocal image of two myelinated nerve fibres within an isolated rat spinal root stained with the Ca²⁺-sensitive dyes Calcium Green-1 and Fura Red. The mean grey value of the paranodal Schwann cell area (selected region of interest indicated in the right panel) and its ratio was used for the analysis. B and C, changes in [Ca²⁺]_i were measured during application of BzATP (150 μM) and UTP (300 μM) into the standard bathing solution and after depletion of intracellular Ca²⁺ stores with cyclopiazonic acid (CPA, 5 μM for 5 min). Note that only the UTP-induced intracellular Ca²⁺ transient was completely and reversibly blocked by CPA.