Gossypol, a component in cottonseed, induced increases in cytosolic Ca2+ levels in Chang liver cells.

The effect of gossypol, a compound found in cottonseed, on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. Gossypol (0.2-5microM) increased [Ca2+](i) in a concentration-dependent manner with an EC(50) value of 1.5microM. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase within 5min after drug application. Removal of extracellular Ca2+ markedly reduced the [Ca2+](i) signals by 80+/-2%. Preincubation with 0.1mM La3+ or 10microM nimodipine abolished the Ca2+ influx. Gossypol (5microM)-induced release of intracellular Ca2+ was reduced by 75% by pretreatment with 1microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+. Conversely, pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. After pretreatment with 5microM gossypol in Ca2+-free medium for several min, addition of 3mM Ca2+ induced a [Ca2+](i) increase of a magnitude nine-fold greater than control. Gossypol (5microM)-induced Ca2+ release was not affected by inhibiting phospholipase C with 2microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Together, this study shows that gossypol induced significant [Ca2+](i) increases in Chang liver cells by releasing Ca2+ from intracellular pools in a phospholipase C-dissociated fashion and by causing La3+- and nimodipine-sensitive Ca2+ influx.

Novel effects of 5,8,11-eicosatriynoic acid, a lipoxygenase inhibitor, on Ca2+ mobilization in Madin Darby canine kidney cells.

The effect of 5,8,11-eicosatriynoic acid, a widely used lipoxygenase inhibitor, on Ca2+ fate in Madin Darby canine kidney cells was examined by using fura-2 as a Ca2+ probe. At concentrations between 2-100 microM 5,8,11-eicosatriynoic acid increased [Ca2+]i concentration-dependently with an EC50 of 20 microM . Extracellular Ca2+ removal decreased the Ca2+ signals, indicating that 5,8,11-eicosatriynoic acid triggered Ca2+ release and Ca2+ influx. 5,8,11 -Eicosatriynoic acid (30 microM) induced a [Ca2+]i increase in Ca2+-free medium after pretreatment with carbonylcyanide m-chlorophenylhydrazone (2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor for 20 min. Conversely, 5,8,11-eicosatriynoic acid pretreatment almost abolished the Ca2+ release induced by carbonylcyanide m-chlorophenylhydrazone and thapsigargin. These results suggest that 30 microM 5,8,11-eicosatriynoic acid released Ca2+ from the endoplasmic reticulum, mitochondria and other stores. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 2-50 microM 5,8,11-eicosatriynoic acid for 10 min. in Ca2+-free medium concentration-dependently. Pretreatment with 10 microM La3+ abolished 30 microM 5,8,11-eicosatriynoic acid -induced [Ca2+]i increases, but adding La3+ during the decay phase had no effect. 5,8,11-Eicosatriynoic acid-induced Ca2+ release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), but was decreased by 60% by 40 microM aristolochic acid. Several other lipoxygenase inhibitors such as baicalein (50 microM), 5.8.11.14-eicosatetraynoic acid (ETYA; 0.1-0.2 mM), caffeic acid (5-50 microM), esculetin (5-50 microM), alpha-pentyl-3-(2-quinolinylmethoxy)-benzenemethanol (REV-5901; 0.1-0.2 mM) and alpha-pentyl-4-(2-quinolinylmethoxy)-benzenemethanol (L-655238; 80-100 microM) had no effect on [Ca2+]i. Collectively, the data suggest that the lipoxygenase inhibitor 5,8,11-eicosatriynoic acid induced a [Ca2+]i increase in renal tubular cells concentration-dependently, by releasing intracellular Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing extracellular Ca2+ influx in a La3+-sensitive manner.

Characteristics and outcome of children with carbon monoxide poisoning with and without smoke exposure referred for hyperbaric oxygen therapy.

OBJECTIVES: To describe the clinical characteristics and outcome of children with carbon monoxide (CO) poisoning with and without smoke exposure referred for hyperbaric oxygen therapy (HBOT), and to determine the association between any of these characteristics and death. SETTING: Regional hyperbaric referral center. PATIENTS: The medical records of 150 children with CO poisoning (COP) who were treated with HBOT between August 92 and September 95 were reviewed. MEASUREMENTS/MAIN RESULTS: COP was defined as a history of probable exposure to CO, with either a carboxyhemoglobin level (COHb) > 25, or COHb < 25 with neurological, respiratory, or cardiac compromise. Major cutaneous burns were described as second degree burns over greater than 20% of the patient's total body surface area (TBSA), or third degree burns over greater than 10% of the patient's TBSA. Children extracted from a closed-space fire who had airway soot, singed facial hair/facial burns, or respiratory distress were defined as having smoke inhalation and carbon monoxide poisoning (CO/SI). CO/SI occurred in 40.1% of patients. Compared to children with COP alone, those with CO/SI were significantly more likely to have a depressed mental status upon arrival to an ED (76.3 % vs 13.6 %, P < 0.001), lower mean initial GCS (6.7 vs 14.7, P < 0.001), lower mean initial pH (7.2 vs 7.4, P < 0.001), respiratory arrest at the scene (68.5% vs 0%, P < 0.001), and cardiac arrest at the scene (25.9% vs 0%, P < 0.001). Children with CO/SI were significantly more likely to have a poor outcome (death) than children with COP alone (22.6% vs. 0%, P < 0.001). Comparing children with CO/SI who died versus survivors, there were significant differences in mean initial COHb (38.3 vs 24.3, P = 0.03), mean initial temperature upon arrival in an ED (94.9 degrees F vs 98.2 degrees, P < 0.006), respiratory arrest at the scene (92% vs 59.6%, P = 0.04), and cardiac arrest at the scene (66.7% vs 13.5%, P < 0.001). Sixty percent of children died who had a combination of risk factors of smoke inhalation, low temperature, high COHb level, and respiratory and cardiac arrest in the field. CONCLUSIONS: These preliminary data suggest that children with COP alone who are treated with HBOT are at low risk for dying regardless of initial COHb level. Children with CO/SI have a significantly higher risk of dying than those children with COP alone. A combination of smoke inhalation, low temperature, high COHb level, respiratory arrest, and cardiac arrest is highly associated with death. Prospective studies are needed to confirm and further define these associations.