Protection of ischemic rat spinal cord white matter: Dual action of KB-R7943 on Na+/Ca2+ exchange and L-type Ca2+ channels
Introduction
Intracellular Ca2+ overload is a common event in the pathophysiology of axonal injury in a variety of disorders including trauma, anoxia and ischemia (for review see Stys, 2004). Reverse operation of the Na+/Ca2+ exchanger (i.e. Na+ efflux, Ca2+ influx mode) is a major contributor to axonal Ca2+ overload and damage observed in these conditions (Stys et al., 1992, Stys and Lopachin, 1997, Imaizumi et al., 1997). Other routes of accumulation have also been suggested including influx through voltage- or transmitter-operated Ca2+-channels (Fern et al., 1995, Li et al., 1999, Li et al., 2000, Agrawal et al., 2000, Brown et al., 2001, Tekkok and Goldberg, 2001) and release from intracellular stores (Stys and Ouardouz, 2002, Thorell et al., 2002, Ouardouz et al., 2003a).
In an in vitro model of rat dorsal column ischemia (combined oxygen glucose deprivation (OGD) for 1 h), we recently observed that removal of extracellular Ca2+ was not protective. Instead, removal of external Ca2+ together with inhibition of release from intracellular stores was required in combination to protect this tissue from ischemia (Ouardouz et al., 2003a). Moreover, we found that a significant portion of the intracellular Ca2+ release was triggered by voltage-dependent gating of L-type Ca2+ channels, which in turn promoted Ca2+ release from ryanodine receptors (Ouardouz et al., 2003a). Therefore, somewhat surprisingly, blocking voltage-gated Ca2+ channels was neuroprotective in this model, but only in the absence of external Ca2+; this was explained by the ability of some Ca2+ channel blockers to interfere with activation of ryanodine receptors in response to depolarization. In this scenario, L-type Ca2+ channels are physically associated with ryanodine receptors on the ER; depolarization will gate the Ca2+ channels and activate ryanodine receptors leading to Ca2+ release from the ER, in a manner very similar to excitation–contraction coupling in skeletal muscle, which does not necessarily require influx of extracellular Ca2+ ions (Franzini-Armstrong and Protasi, 1997). The effect of an inhibitor of Na+/Ca2+-exchange ((2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), KB-R7943) was tested in this model. Unexpectedly, KB-R7943 showed a very protective effect both in the presence and absence of external Ca2+. This result could not be explained solely on the basis of selective blockade of Ca2+ influx through the Na+/Ca2+-exchanger, as removal of extracellular Ca2+ itself was not protective. We hypothesized that KB-R7943 might additionally act on L-type Ca2+ channels, thereby interfering with release from intracellular stores which we have shown to play a major role in ischemic white matter injury. These results have been published in abstract form (Ouardouz et al., 2003b).
Section snippets
Recordings of propagated CAPs in dorsal columns
Experiments were performed on spinal cord dorsal columns in vitro from adult Long Evans male rat (250–350 g) (for details see Li et al., 1999). Rats were deeply anesthetized with sodium pentobarbital, and then perfused intra-aortically with a cold zero-Ca2+ solution containing (in mM): NaCl 126, KCl 3, MgSO4 2, NaHCO3 26, NaH2PO4 1.25, MgCl2 2, dextrose 10 and EGTA 0.5 mM, oxygenated with 95% O2–5% CO2 mixture. Thoracic spinal cord was removed and placed in cold oxygenated zero-Ca2+ solution. One
Protective effect of KB-R7943 in dorsal column ischemia
Combined oxygen and glucose deprivation (OGD) at 37 °C for 1 h, caused severe injury to spinal cord dorsal columns as assessed by irreversible and near-complete failure of CAP propagation (measured 3 h after OGD), which could not be rescued by removal of bath Ca2+ (+EGTA) (Fig. 1A and B). The injury that occurs in zero-external Ca2+ is mediated in large part by release of intracellular Ca2+ stores triggered by L-type Ca2+ channel activation of ryanodine receptors (Ouardouz et al., 2003a).
KB-R7943
Discussion
KB-R7943 has been used as a selective inhibitor of the reverse (i.e. Ca2+-import) mode of the Na+/Ca2+ exchanger in many preparations to study the involvement of this ion transporter in a variety of pathological and physiological situations including white matter injury (for review see Stys, 2004), grey matter injury (Schröder et al., 1999), cardiac ischemia (Baczko et al., 2003, Seki et al., 2002), excitotoxicity (Czyz˙ and Kiedrowski, 2002, Kiedrowski et al., 2004), and synaptic transmission (
Acknowledgements
Supported by NINDS R01 NS40087-01 (operating), and Heart and Stroke Foundation of Ontario Career Investigator Award (salary) grants to PKS; Premier's Research Excellence Award, Province of Ontario (MO); Grant in Aid to GWZ from the Heart and Stroke Foundation of Alberta, the Northwest Territories and Nunavut. GWZ is a Senior Scholar of the Alberta Heritage Foundation of Medical Research and a Canada Research Chair in Molecular Neurobiology; and NINDS R29 NS37390 to LK.
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