Alterations in Phosphatidylcholine Metabolism of Stretch‐Injured Cultured Rat Astrocytes

Abstract

The primary objective of this study was to determine the influence of stretch-induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silasticbottomed tissue culture wells in medium that was usually supplemented with 10~M unlabeled arachidonate. Cell injury was produced by stretching (5-10 mm) the Silastic membrane with a 50-ms pulse of compressed air. Stretch-induced cell injury increased the incorporation of [ 3H]cholineinto PC in an incubation time-and stretch magnitude-dependent manner. PC biosynthesis was increased three-to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch-induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A 2 (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch-induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretreating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch-dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n -acetylcysteine). Arachidonate-enriched cells were more susceptible to stretch-induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non-arachidonate-enriched cells. In summary, the data suggest that stretch-induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron-catalyzed Haber-Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cell's membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch-induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch-induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction. Key Words: Cultured astrocytes-Stretch-induced cell injury-Phosphatidylcholine biosynthesis-Phospholipase activity-Phosphocholine cytidylyltransferase activity-Oxyradical scavengers-Antioxidants.

Antagonism of Group I Metabotropic Glutamate Receptors and PLC Attenuates Increases in Inositol Trisphosphate and Reduces Reactive Gliosis in Strain-Injured Astrocytes

“…We have validated our model by demonstrating that in vitro stretch injury reproduces many of the post-traumatic responses observed in vivo, including intracellular lesions to the mitochondria, Golgi, and cytoskeletal elements in astrocytes and neurons (Dietrich et al, 1994;Ellis et al, 1995;McKinney et al, 1996), increases in astrocyte and neuronal permeability (Pettus et al, 1994;Ellis et al, 1995;Weber et al, 1999), phospholipase activation (Wei et al, 1982;Lamb et al, 1997), free radical and isoprostane formation (McKinney et al, 1996;Lamb et al, 1997;Hoffman et al, 2000), and decreases in astrocyte and neuronal mitochondrial membrane potential and ATP levels (Ahmed et al, 2000;Vagnozzi et al, 1999). Stretch injury of cultured neurons also decreases the Mg 21 block of the NMDA receptor (Zhang et al, 1996), and causes a novel stretch-induced delayed neuronal depolarization, which may be related to transient neuronal dysfunction observed in vivo (Hamm et al, 1993;Tavalin et al, 1995), and also increases activation of the AMPA receptor by decreasing AMPA receptor desensitization (Goforth et al, 1999).…”

Protein kinase signaling cascades in CNS trauma

“…The range of membrane displacements generated in this model correspond to biaxial strains, or stretch, that are relevant to those that occur in humans after rotational acceleration-deceleration injury. This model replicates many of the post-traumatic responses observed in vivo, including transient increases in intracellular calcium (41), activation of phospholipases (42,43), free radical formation (44), and reduction and release of intracellular ATP (45). Our laboratory has utilized this model to study the activation of protein kinase cascades by mechanical strain, and the factors that initiate their activation, in primary cultures of rat cortical astrocytes.…”

Traumatic injury of cultured astrocytes alters inositol (1,4,5)-trisphosphate-mediated signaling

“…To better understand the pathophysiological processes involved in TBI, our laboratory has developed an in vitro model of trauma that produces tissue strain or "stretch" . We have validated our model by demonstrating that in vitro stretch injury produces many of the posttraumatic responses observed in vivo, including intracellular lesions to the mitochondria, Golgi, and cytoskeletal elements in astrocytes and neurons (Dietrich et al, 1994;Ellis et al, 1995;McKinney et al, 1996), increases in astrocyte and neuronal permeability (Pettus et al, 1994;Ellis et al, 1995), phospholipase activation (Wei et al, 1982;Lamb et al, 1997), and free radical and isoprostane formation (McKinney et al, 1996, Lamb et al, 1997Hoffman et al, 2000). Stretch injury of cultured neurons also produces a novel stretch-induced delayed neuronal depolarization that may be related to transient neuronal dysfunction observed in vivo (Hamm et al, 1994;Tavalin et al, 1995), and also increases activation of the 3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor by decreasing AMPA receptor desensitization .…”