The regional distribution of cholesterol, phospholipid and protein content was determined on pools of human lenses ranging from 13 to 68 years old. The study was undertaken to establish age matched controls for comparison with cataractous lenses. Future spectroscopic structure analysis of human lenses will be performed and the results related to chemical composition. The molar cholesterol to phospholipid ratio was 3.5 +/- 0.3 for human lens. This ratio is high for human tissue. The lens ratio increased from 2.2 +/- 0.3 in the equatorial region to 9.2 +/- 1.6 in the nuclear region. This trend was also observed in the bovine lens. The relative amount of protein increased concomitantly from 0.13 +/- 0.02 Kg protein per gram lipid in the equatorial region to 0.33 +/- 0.06 in the nucleus. The cholesterol to protein ratio remained constant throughout the lens at 0.073 +/- 0.003 Kg suggesting cholesterol could be associated with the crystallin proteins. In partially purified membrane preparations the cholesterol to phospholipid molar ratio was 2.6 +/- 0.2 and 3.2 +/- 0.2 for the cortex and nucleus respectively, three times lower than for the whole tissue. The high cholesterol content could account for the observed rigidity of membranes measured by infrared spectroscopic examination of the CH stretching band.
Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.
The influence of vanadate upon the adrenergic response of the rabbit iris (dilation) was examined in vivo, while the effects of vanadate upon adrenergic responses of the rabbit ileum and guinea pig vas deferens were investigated in vitro. Intravenously administered vanadate (2 mg/Kg) sensitized the iris to topically administered epinephrine; a small quantity of epinephrine, that elicited no change of pupil size following topical administration to the eye in the normal rabbit, produced a marked pupillary dilation in animals treated systemically with vanadate. The response of the isolated ileum to vanadate (1-10 micrograms) was identical to that elicited by norepinephrine or epinephrine. Brief exposure of the ileum to vanadate, norepinephrine, or epinephrine resulted in a transient cessation of rhythmic activity and reduction of mean tension. When the ileum was superfused continuously with solutions containing vanadate (10 micrograms/ml; 5.4 X 10(-5) M), the rhythmic activity and mean tension returned to control values within several minutes, suggesting tachyphylaxis. Exposure of the guinea pig vas deferens to vanadate did not elicit a contractile response. However, when the vas deferens was superfused continuously (greater than 60 min) with vanadate (5.4 X 10(-5) M), both the amplitude and duration of the contraction elicited by epinephrine were increased. Consistent with the above findings is the hypothesis that vanadate might inhibit the mechanisms responsible for the removal of epinephrine or norepinephrine from the site of action within a tissue.
Elevated intraocular pressure is associated with glaucomatous optic nerve damage. Other investigators have shown functional changes in optic nerve head astrocytes subjected to elevated hydrostatic pressure (HP) for 1 to 5 days. Recently, the authors reported ERK1/2, p90(RSK) and NHE1 phosphorylation after 2 hours. Here they examine calcium responses at the onset of HP to determine what precedes ERK1/2 phosphorylation.
