Jerry S. McKinney

Stretch-Induced Injury of Cultured Neuronal, Glial, and Endothelial Cells Effect of Polyethylene Glycol–Conjugated Superoxide Dismutase

BACKGROUND AND PURPOSE There is abundant evidence that after in vivo traumatic brain injury, oxygen radicals contribute to changes in cerebrovascular structure and function; however, the cellular source of these oxygen radicals is not clear. The purpose of these experiments was to use a newly developed in vitro tissue culture model to elucidate the effect of strain, or stretch, on neuronal, glial, and endothelial cells and to determine the effect of the free radical scavenger polyethylene glycol-conjugated superoxide dismutase (PEG-SOD; pegorgotein, Dismutec) on the response of each cell type to trauma. METHODS Rat brain astrocytes, neuronal plus glial cells, and aortic endothelial cells were grown in cell culture wells with 2-mm-thick silastic membrane bottoms. A controllable, 50-millisecond pressure pulse was used to transiently deform the silastic membrane and thus stretch the cells. Injury was assessed by quantifying the number of cells that took up the normally cell-impermeable dye propidium iodide. Some cultures were pretreated with 100 to 300 U/mL PEG-SOD. RESULTS Increasing degrees of deformation produced increased cell injury in astrocytes, neuronal plus glial cultures, and aortic endothelial cells. By 24 hours after injury, all cultures showed evidence of repair as demonstrated by cells regaining their capacity to exclude propidium iodide. Compared with astrocytes or neuronal plus glial cultures, endothelial cells were much more resistant to stretch-induced injury and more quickly regained their capacity to exclude propidium iodide. PEG-SOD had no effect on the neuronal or glial response to injury but reduced immediate posttraumatic endothelial cell dye uptake by 51%. CONCLUSIONS These studies further document the utility of the model for studying cell injury and repair and further support the vascular endothelial cell as a site of free radical generation and radical-mediated injury. On the assumption that, like aortic endothelial cells, stretch-injured cerebral endothelial cells also produce oxygen radicals, our results further suggest the endothelial cell as a site of therapeutic action of free radical scavengers after traumatic brain injury.

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 Silastic‐bottomed tissue culture wells in medium that was usually supplemented with 10 µM 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]choline into 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 A2 (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 pretrating (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 cells 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.

Effect of Ca2+ on in vitro astrocyte injury

Abstract: Current literature suggests that a massive influx of Ca2+ into the cells of the CNS induces cell damage associated with traumatic brain injury (TBI). Using an in vitro model for stretch‐induced cell injury developed by our laboratory, we have investigated the role of extracellular Ca2+ in astrocyte injury. The degree of injury was assessed by measurement of propidium iodide uptake and release of lactate dehydrogenase. Based on results of in vivo models of TBI developed by others, our initial hypothesis was that decreasing extracellular Ca2+ would result in a reduction in astrocyte injury. Quite unexpectedly, our results indicate that decreasing extracellular Ca2+ to levels observed after in vivo TBI increased astrocyte injury. Elevating the extracellular Ca2+ content to twofold above physiological levels (2 mM) produced a reduction in cell injury. The reduction in injury afforded by Ca2+ could not be mimicked with Ba2+, Mn2+, Zn2+, or Mg2+, suggesting that a Ca2+‐specific mechanism is involved. Using 45Ca2+, we demonstrate that injury induces a rapid influx of extracellular Ca2+ into the astrocyte, achieving an elevation in total cell‐associated Ca2+ content two‐ to threefold above basal levels. Pharmacological elevation of intracellular Ca2+ levels with the Ca2+ ionophore A23187 or thapsigargin before injury dramatically reduced astrocyte injury. Our data suggest that, contrary to popular assumptions, an elevation of total cell‐associated Ca2+ reduces astrocyte injury produced by a traumatic insult.

14,15-Epoxyeicosatrienoic acid inhibits platelet aggregation in mouse cerebral arterioles.

Background and Purpose: Epoxygenase metabolites of arachidonic acid are produced by several tissues and have been shown to inhibit in vitro platelet aggregation. The purpose of the present investigation was to determine whether 14,15- or 8,9-epoxyeicosatrienoic acid, epoxygenase derivatives of arachidonic acid, affect the speed of platelet aggregation in in vivo mouse cerebral arterioles. Methods: We performed a craniectomy in 116 anesthetized male mice and observed the pial arterioles by microscopy. We induced in situ platelet aggregation using a mercury light and intravascularly injected fluorescein dye. Results: Indomethacin (0.5 mg/kg i.p.), a known cyclooxygenase inhibitor, and 14,15- epoxyeicosatrienoic acid (0.3 mg/kg i.v.) increased the time necessary for the light plus dye to induce the first arterial platelet aggregate by 35% and 26%, respectively, whereas 8,9- epoxyeicosatrienoic acid (0.3 mg/kg i.v.) had no effect. Analysis of mouse serum by radioimmunoassay showed that the degree of inhibition of platelet aggregation by indomethacin and epoxyeicosatrienoic acids correlated with the degree of inhibition of thromboxane production. Conclusions: We conclude that 14,15-epoxyeicosatrienoic acid is a potent inhibitor of in vivo platelet aggregation but cannot conclusively confirm that its effect on aggregation occurs via its reduction of platelet thromboxane A2. Because epoxyeicosatrienoic acids are produced by several tissues, including brain and vascular tissue, they may be important in vivo modulators of platelet aggregation and hemostasis.

Brain and tissue distribution of polyethylene glycol-conjugated superoxide dismutase in rats.

Background and Purpose The purpose of this study was to determine the distribution of polyethylene glycol–conjugated superoxide dismutase in the brain, cerebrospinal fluid, and various organs. Methods Distribution of iodine-125–labeled polyethylene glycol–conjugated superoxide dismutase was determined in three groups of male Sprague-Dawley rats: a normotensive sham control group (n=9) and groups given 125I-labeled polyethylene glycol-conjugated superoxide dismutase either 30 minutes before n=10) or 30 minutes after (n=7) norepinephrine-induced hypertensive injury. Results In the first 30 minutes after intravenous administration, polyethylene glycol–conjugated superoxide dismutase plasma activity declined to 70% of the initial value and then decreased negligibly between 30 and 90 minutes. Levels of 125l-labeled polyethylene glycol–conjugated superoxide dismutase in normotensive animals were low in the brain and cerebrospinal fluid and highest in kidney. Brain levels of polyethylene glycol–conjugated superoxide dismutase were elevated only in those rats that received it before hypertensive injury, however, cerebrospinal fluid levels were elevated in animals receiving the drug either before or after hypertensive injury. Conclusion Our results suggest that the blood–brain barrier becomes more permeable to polyethylene glycol–conjugated superoxide dismutase only during the hypertensive period but that the bloodcerebrospinal fluid barrier sustains more permanent injury. We suggest that the therapeutic effectiveness of polyethylene glycol–conjugated superoxide dismutase in hypertensive brain injury is due to its action in the vascular wall or to its extracellular activity in the cerebrospinal fluid.

Inositol 1,4,5-trisphosphate may be a signal for f-Met-Leu-Phe-induced intracellular Ca mobilisation in human leucocytes (HL-60 cells)

Pemeabilised, dimethyl sulphoxide‐differentiated HL‐60 human myelomonocytic leukemia cells accumulate 45Ca in an ATP‐dependent manner. The 45Ca is taken up by a pool thought to be a component of the endoplasmic reticulum. Inositol trisphosphate induced a rapid release of Ca from this pool, suggesting that this molecule which is formed in these cells in response to f‐Met‐Leu‐Phe may play a role in agonist‐induced Ca metabolism.

Enhancement of cyclic AMP modulated salivary amylase secretion by protein kinase C activators

The effect of protein kinase C activators on isoproterenol-induced amylase secretion were investigated in isolated rat parotid cells. Pretreatment with phorbol dibutyrate potentiated isoproterenol-induced amylase secretion. This effect of phorbol dibutyrate was mimicked by dioctanoylglycerol or carbachol. Phorbol dibutyrate also potentiated secretion evoked by the adenylate cyclase activator forskolin and by dibutyryl cAMP. Neither phorbol dibutyrate nor carbachol enhanced isoproterenol-induced cAMP accumulation. The present study reveals a coordinate interaction between cAMP and protein kinase C at a step in the secretory mechanism distal to cAMP generation.

Calium, prostaglandins and the phosphatidylinositol effect in exocrine gland cells

Abstract The hypothesis that arachidonic acid metabolism might be involved in Ca-mobilization mechanisms in exocrine gland cells was investigated. Arachidonate (10−4M) failed to stimulate protein secretion from slices of pancreas, parotid or lacrimal glands and failed to stimulate 86Rb efflux from parotid or lacrimal glands. The stimulation of protein secretion (all three glands) or 86Rb efflux (parotid and lacrimal glands) by appropriate secretagogues was unaffected by 10−5M indomethacin. Eicosatetraynoic acid (2×10−5M) inhibited 86Rb efflux due to carbachol but not that due to physalaemin or ionomycin. Nordihydroguaiaretic acid inhibited lacrimal and parotid gland responses only at high (10−4M) concentration. Collectively, these results argue against an obligatory role for arachidonate metabolites in Ca-mediated responses of these exocrine glands. In the exocrine glands activation by neurotransmitters (or analogs) of receptors that mobilize cellular Ca also stimulates the incorporation of 32PO4 into phosphatidylinositol (1–3). Michell (4,5) has suggested that in some manner this alteration in phospholipid metabolism may be functionally responsible for the opening of surface membrane Ca gates which presumably precedes the expression of a number of Ca-mediated responses by the exocrine cell. That this reaction probably preceeds Ca mobilization is deduced primarily from two experimental observations. First, receptor activation of phosphatidylinositol turnover is not prevented by Ca omission (6–8). Second, the effect is not mimicked by the divalent cationophore A-23187, while other effects of receptor activation are mimicked by this compound (7–9). There has also been some speculation as to the manner in which altered phosphatidylinositol metabolism might be involved in the Ca-gating mechanism (10–14). One such hypothesis suggests that receptor activation may lead to phosphatidylinositol breakdown which in turn leads to the release of free arachidonate (13, 14). As free arachidonate is generally believed to be the rate-limiting substrate for prostaglandin synthesis (15), the resulting prostaglandins might act to mobilize Ca or might act in concert with Ca (13, 14). There is evidence for this hypothesis for the mouse pancreas, where exogenous arachidonate and prostaglandins can stimulate amylase release (13). The effects of arachidonate, carbachol, caerulein and pancreozmin were all antagonized by sub-micromolar concentrations of indomethacin (13), a potent cyclooxygenase inhibitor (15). Additionally, recent reports have demonstrated stimulation by acetylcholine of prostaglandin E synthesis in mouse pancreas (16, 17). The purpose of this study was to examine the general applicability of this hypothesis by investigating the effects of arachidonate and substances that inhibit prostaglandin formation in two other exocrine tissues that show a prominent phosphatidylinositol turnover — the rat parotid and lacrimal glands.

The effect of dietary n-3 fatty acids on in vivo platelet aggregation in the cerebral microcirculation.

Diets enriched in n-3 fish oil have been suggested to decrease coronary artery disease in part through their ability to decrease cyclooxygenase-dependent platelet aggregation. However little is known concerning the effect of n-3 fatty acids on in vivo platelet aggregation. The purpose of these experiments was to determine whether dietary n-3 fatty acids affect the rate at which platelet aggregation occurs in cerebral arterioles. Fish oil (200 mg eicosapentaenoic acid + 143 mg docosahexaenoic acid/kg), corn oil or water was given daily by gavage to mice (n = 30) for six weeks and then in vivo platelet aggregation was induced by the light plus dye method, which injuries the endothelium. Two additional groups of mice were acutely treated with saline or indomethacin (0.5 mg/kg, ip), with the latter serving as a positive control for therapeutic inhibition of platelet aggregation. Serum thromboxane B2 was analyzed by RIA. All fed groups gained weight equally. Serum thromboxane B2 was decreased by 40% in the fish oil group (p = 0.05 vs. corn oil, p = 0.07 vs. water). The mean (+/- SE) time to first aggregate in pial arterioles was 101 +/- 6, 91 +/- 6 and 101 +/- 9 seconds in the fish corn oil and water groups, respectively. Indomethacin significantly increased the time to first arteriolar aggregate by 35% (p < 0.002) and caused an 80% reduction in serum thromboxane. These studies show dietary fish oil produces a moderate reduction in serum TxB2 level and does not affect arteriolar platelet aggregation whereas indomethacin produces a drastic TxB2 reduction and significantly slows platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation by diacylglycerol of calcium-evoked amylase secretion from intact and permeabilized pancreatic acinar cells

The role of diacylglycerol in the mechanism of amylase release was investigated in isolated rat pancreatic acinar cells. Carbachol produced a time-dependent and dose-related increase in diacylglycerol production which paralleled the time course of amylase secretion. The addition of atropine to acinar cells pretreated with 100 microM carbachol produced a lag in the fall in diacylglycerol levels, which was preceded by a prompt fall in cytosolic Ca2+ and amylase secretion. A threshold concentration of ionomycin amplified the modest action of dioctanoylglycerol on amylase secretion. Ca2(+)-evoked amylase release elicited by saponin permeabilized acinar cells was markedly enhanced by dioctanoylglycerol. These collective findings support the hypothesis that diacylglycerol alone is not an adequate messenger to mediate pancreatic amylase release, but does serve to modulate the actions of Ca2+.