The Na+/Ca2+ exchangers, NCX and NCKX, contribute to the synergistic [Ca2+]i overload produced by concurrent ischemia and acidosis in neurons

Abstract

A major contributing factor to the expansion of the core region following ischemic stroke is sustained elevations of intracellular Ca2+ ([Ca2+]i) in neurons in the penumbral region. Our laboratory previously showed that ischemia and acidosis, which occur concurrently during ischemic stroke, produce synergistic increases in neuronal [Ca2+]i that result in neuronal death. This synergistic [Ca2+]i overload is in part due to activation of acid‐sensing ion channel 1a (ASIC1a). However, the inhibition of ASIC1a alone did not abolish the synergistic potentiation, and thus other molecular mechanisms appear to be involved. Cultured cortical neurons from E18 rats were subjected to acute chemical ischemia (glucose‐free saline solution + 4 mM azide) and/or acidosis (pH 6.0) followed by reperfusion. Responses were studied using fluorometric Ca2+ measurements and whole‐cell patch‐clamp recording techniques. Ischemia + acidosis (2 min) evoked a rapid, transient increase in [Ca2+]i in the neurons, followed by a slow rise in [Ca2+]i. Upon washout of the ischemia + acidosis solution, the [Ca2+]i rebounds to a second, slower decaying peak before returning to baseline levels. The [Ca2+]i increases produced by ischemia + acidosis were significantly greater than the sum of the [Ca2+]i increases produced by ischemia or acidosis alone. Application of ischemia + acidosis to neurons under current‐clamp mode produces a large transient depolarization of the membrane potential from −53 ± 1 mV to −12 ± 1 mV, which repolarizes to −41 ± 1 mV during ischemia + acidosis, and completely repolarizes upon washout. Neurons held under voltage‐clamp (−70 mV) respond to ischemia + acidosis with large (−1112 ± 165 nA) transient inward currents that decay to sustained levels (−96 ± 14 pA). Net charge influx was synergistically amplified by ischemia + acidosis, when compared to the sum of ischemia and acidosis alone. However, membrane potential changes were not synergistically enhanced. Both transient components of the [Ca2+]i increases (initial and reperfusion‐associated) were eliminated when Ca2+ was removed from the external solution. The Na+/Ca2+ exchangers, NCX and NCKX, were implicated as sources of Ca2+ influx by inhibition with benzamil and YM244769, and by changes to [Ca2+]i responses produced when external [K+] was altered. Ca2+ release from the endoplasmic reticulum does not contribute to the linear rise in [Ca2+]i in the presence of the ischemia + acidosis solution as neither cyclopiazonic acid nor caffeine pretreatment effected this phase of the response. Similarly, the mitochondrial Na+/Ca2+ exchanger, NCLX, was found not to contribute to this phase of the [Ca2+]i response, as CGP37157 did not inhibit [Ca2+]i increases. Taken together, our data suggest that NCX and NCKX, acting in Ca2+ influx mode, contribute to the synergistic potentiation of [Ca2+]i overload produced by ischemia + acidosis. Thus, activation of these proteins during ischemia and reperfusion is likely to exacerbate ischemic stroke injury.Support or Funding InformationAmerican Heart Association Grant‐In‐Aid, 17GRNT33661273 (JC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.