Acidosis - Hypoxia - Astrocyte energetics, function, and death under conditions of incomplete ischemia: A mechanism of glial death in the penumbra

Cerebral artery occlusion produces regions of incomplete ischemia (the ischemic penumbra), which, in the absence of reflow, undergo progressive metabolic deterioration culminating in infarction. The factors causing infarction are not yet established, but progression to cell death is preceded by progressive acidosis, decreasing glucose utilization, and ATP depletion.

To identify potential mechanisms of glial death in the ischemic penumbra, astrocytes in culture were subjected to conditions that occur during incomplete ischemia: hypoxia, acidosis, and raised extracellular K+. Neither acidosis (to pH 6.2) nor chemical hypoxia (5 mM azide) alone produced significant astrocyte death or marked ATP depletion.

By contrast, hypoxia combined with acidosis caused near-complete ATP depletion by 3.5 h and 70% cell death after 7 h. Glycolytic rate increased during hypoxia alone but decreased during hypoxia with acidosis. Since glycolysis is the sole source of ATP production during hypoxia, acidosis inhibition of glycolysis is a likely cause of the far greater ATP depletion resulting from hypoxia with acidosis. Glutamate uptake was reduced during hypoxia and further reduced during hypoxia with acidosis, consistent with the changes in astrocyte ATP.

Glutamate uptake, ATP levels, and glycolytic rate each exhibited reductions that were progressive over 3 h of hypoxia with acidosis, and these changes were accompanied by progressive intracellular acidosis. Since ATP depletion leads to acidosis, and acidosis inhibits glycolysis, these findings suggest a regenerative cycle initiated by the combination of hypoxia with acidosis. This cycle could result in progressive metabolic decline and cell death in the ischemic penumbra.  GLIA 21:142–153, 1997. © 1997 Wiley-Liss, Inc.

Acidosis - Hypoxia - A Unique Pathway of Cardiac Myocyte Death Caused By Hypoxia-Acidosis

Chronic hypoxia in the presence of high glucose leads to progressive acidosis of cardiac myocytes in culture. The condition parallels myocardial ischemia in vivo, where ischemic tissue becomes rapidly hypoxic and acidotic. Cardiac myocytes are resistant to chronic hypoxia at neutral pH but undergo extensive death when the extracellular pH (pH[o]) drops below 6.5.

A microarray analysis of 20 000 genes (cDNAs and expressed sequence tags) screened with cDNAs from aerobic and hypoxic cardiac myocytes identified> 100 genes that were induced by >2-fold and ∼20 genes that were induced by >5-fold. One of the most strongly induced transcripts was identified as the gene encoding the pro-apoptotic Bcl-2 family member BNIP3.

Northern and western blot analyses confirmed that BNIP3 was induced by 12-fold (mRNA) and 6-fold (protein) during 24 h of hypoxia. BNIP3 protein, but not the mRNA, accumulated 3.5-fold more rapidly under hypoxia–acidosis. Cell fractionation experiments indicated that BNIP3 was loosely bound to mitochondria under conditions of neutral hypoxia but was translocated into the membrane when the myocytes were acidotic. Translocation of BNIP3 coincided with opening of the mitochondrial permeability pore (MPTP).

Paradoxically, mitochondrial pore opening did not promote caspase activation, and broad-range caspase inhibitors do not block this cell death pathway. The pathway was blocked by antisense BNIP3 oligonucleotides and MPTP inhibitors. Therefore, cardiac myocyte death during hypoxia–acidosis involves two distinct steps: (1) hypoxia activates transcription of the death-promoting BNIP3 gene through a hypoxia-inducible factor-1 (HIF-1) site in the promoter and (2) acidosis activates BNIP3 by promoting membrane translocation. This is an atypical programmed death pathway involving a combination of the features of apoptosis and necrosis.

In this article, we will review the evidence for this unique pathway of cell death and discuss its relevance to ischemic heart disease. The article also contains new evidence that chronic hypoxia at neutral pH does not promote apoptosis or activate caspases in neonatal cardiac myocytes.

Acidosis - Hypoxia - Rapid astrocyte death induced by transient hypoxia, acidosis, and extracellular ion shifts

Death of astrocytes requires hours to days in injury models that use hypoxia, acidosis, or calcium paradox protocols. These methods do not incorporate the shifts in extracellular K+, Na+, Cl−, and Ca2+ that accompany acute brain insults. We studied astrocyte survival after exposure to hypoxic, acidic, ion-shifted Ringer (HAIR), with respective [Ca2+], [K+], [Na+], [Cl−], and [HCO] of 0.13, 65, 51, 75, and 13 mM (15% CO2/85% N2, pH 6.6).

Intracellular pH (pHi) was monitored with the fluorescent dye BCECF. Cell death was indicated by a steep fall in the pH-insensitive, 440-nm-induced fluorescence (F440) and was confirmed by propidium iodide staining. After 15–40-min HAIR exposure, reperfusion with standard Ringer caused death of most cultured (and acutely dissociated) astrocytes within 20 min. Cell death was not prevented if low Ca2+ was maintained during reperfusion. Survival fell with increased HAIR duration, elevated temperature, or absence of external glucose.

Comparable durations of hypoxia, acidosis, or ion shifts alone did not lead to acute cell death, while modest loss was noted when acidosis was paired with either hypoxia or ion shifts. Severe cell loss required the triad of hypoxia, acidosis, and ion shifts. Intracellular pH was significantly higher in HAIR media, compared with solutions of low pH alone or with low pH plus hypoxia.

These results indicate that astrocytes can be killed rapidly by changes in the extracellular microenvironment that occur in settings of traumatic and ischemic brain injury. GLIA 34:134–142, 2001. © 2001 Wiley-Liss, Inc.

Acidosis - Hypoxia - Ischemia: from Acidosis to Oxidation

Organ damage can occur quickly when blood flow is compromised. Lactic acidosis has long been associated with such ischemia, and many physicians assume that organ damage is caused by this acidosis. However, reviewing the literature related to hypoxia and ischemia reveals little data to support the concept of acidosis as damaging to tissue. In contrast, recent studies indicate that the acidosis is actually protective, even during reperfusion when cellular damage may occur.

Reperfusion is accompanied by generation of free radicals and other reactive species that can damage proteins, membranes, and nucleic acids, supporting an emerging view that implicates these reactive species in the actual tissue damage. The critical targets of the damaging species are not known, but reaction with key enzymes and structural proteins could certainly disrupt organ function.

Cellular proteins are oxidatively modified during reperfusion, in part by metal- catalyzed oxidation in which cellular iron plays a key role. Metal- catalyzed oxidation of proteins may be important in the pathogenesis of other disorders, including the potentially blinding disease, retinopathy of the premature.